Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

ABSTRACT

The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/235,409 filed Aug. 12, 2016, now U.S. Pat. No. 10,017,755, which is adivisional of U.S. patent application Ser. No. 14/875,352 filed Oct. 5,2015, now U.S. Pat. No. 9,422,537, which is a divisional of U.S. patentapplication Ser. No. 13/992,003, now U.S. Pat. No. 9,157,075, which is a35 U.S.C. 371 national application of PCT/US2012/22671 filed Jan. 26,2012, which claims priority or the benefit under 35 U.S.C. 119 ofEuropean Application No. 11152252.0 filed Jan. 26, 2011, U.S.Provisional Application No. 61/483,116 filed May 6, 2011, and U.S.Provisional Application No. 61/546,602 filed Oct. 13, 2011. The contentsof these applications are fully incorporated herein by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made in part with Government support underCooperative Agreement DE-FC36-08GO18080 awarded by the Department ofEnergy. The government has certain rights in this invention.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to polypeptides having cellobiohydrolaseactivity and polynucleotides encoding the polypeptides. The inventionalso relates to nucleic acid constructs, vectors, and host cellscomprising the polynucleotides as well as methods of producing and usingthe polypeptides.

Description of the Related Art

Cellulose is a polymer of the simple sugar glucose covalently linked bybeta-1,4-bonds. Many microorganisms produce enzymes that hydrolyzebeta-linked glucans. These enzymes include endoglucanases,cellobiohydrolases, and beta-glucosidases. Endoglucanases digest thecellulose polymer at random locations, opening it to attack bycellobiohydrolases. Cellobiohydrolases sequentially release molecules ofcellobiose from the ends of the cellulose polymer. Cellobiose is awater-soluble beta-1,4-linked dimer of glucose. Beta-glucosidaseshydrolyze cellobiose to glucose.

The conversion of lignocellulosic feedstocks into ethanol has theadvantages of the ready availability of large amounts of feedstock, thedesirability of avoiding burning or land filling the materials, and thecleanliness of the ethanol fuel. Wood, agricultural residues, herbaceouscrops, and municipal solid wastes have been considered as feedstocks forethanol production. These materials primarily consist of cellulose,hemicellulose, and lignin. Once the lignocellulose is converted tofermentable sugars, e.g., glucose, the fermentable sugars are easilyfermented by yeast into ethanol.

The present invention provides polypeptides having cellobiohydrolaseactivity and polynucleotides encoding the polypeptides.

The T. byssochlamydoides GH7 polypeptide having cellobiohydrolaseactivity (SEQ ID NO: 2) shares 87.41% identity (excluding gaps) to thededuced amino acid sequence of an cellobiohydrolase from Talaromycesemersonii (GENESEQP: AYL28232).

The T. byssochlamydoides GH7 polypeptide having cellobiohydrolaseactivity (SEQ ID NO: 4) shares 78.94% identity (excluding gaps) to thededuced amino acid sequence of an glycosyl hydrolase family 7 proteinfrom Neosartorya fischeri (SWISSPROT:A1DAP8).

SUMMARY OF THE INVENTION

The present invention relates to isolated polypeptides havingcellobiohydrolase activity selected from the group consisting of:

(a) a polypeptide having at least 90% sequence identity to the maturepolypeptide of SEQ ID NO: 2 or a polypeptide having at least 80%sequence identity to the mature polypeptide of SEQ ID NO: 4;

(b) a polypeptide encoded by a polynucleotide having at least 90%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 or the cDNA sequence thereof or a polypeptide encoded by apolynucleotide having at least 80% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 3 or the cDNA sequencethereof;

(c) a variant comprising a substitution, deletion, and/or insertion ofone or more (several) amino acids of the mature polypeptide of SEQ IDNO: 2 or of SEQ ID NO: 4; and

(d) a fragment of the polypeptide of (a), (b), or (c) that hascellobiohydrolase activity.

The present invention also relates to isolated polypeptides comprising acatalytic domain selected from the group consisting of:

(a) a catalytic domain having at least 90% sequence identity to thecatalytic domain of SEQ ID NO: 2 or a catalytic domain having at least80% sequence identity to the catalytic domain of SEQ ID NO: 4;

(b) a catalytic domain encoded by a polynucleotide having at least 90%sequence identity to the catalytic domain coding sequence of SEQ ID NO:1 or a catalytic domain encoded by a polynucleotide having at least 80%sequence identity to the catalytic domain coding sequence of SEQ ID NO:3;

(c) a variant of a catalytic domain comprising a substitution, deletion,and/or insertion of one or more (several) amino acids of the catalyticdomain of SEQ ID NO: 2 or of SEQ ID NO: 4; and

(d) a fragment of a catalytic domain of (a), (b), or (c), which hascellobiohydrolase activity.

The present invention also relates to an enzyme composition comprisingthe polypeptide of the invention; isolated polynucleotides encoding thepolypeptides of the present invention; nucleic acid constructs,recombinant expression vectors, and recombinant host cells comprisingthe polynucleotides; and methods of producing the polypeptides.

The present invention also relates to methods for degrading orconverting a cellulosic material, comprising: treating the cellulosicmaterial with an enzyme composition in the presence of a polypeptidehaving cellobiohydrolase activity of the present invention. In oneaspect, the method further comprises recovering the degraded orconverted cellulosic material.

The present invention also relates to methods of producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with an enzyme composition in the presence of a polypeptidehaving cellobiohydrolase activity of the present invention; (b)fermenting the saccharified cellulosic material with one or more(several) fermenting microorganisms to produce the fermentation product;and (c) recovering the fermentation product from the fermentation.

The present invention also relates to methods of fermenting a cellulosicmaterial, comprising: fermenting the cellulosic material with one ormore (several) fermenting microorganisms, wherein the cellulosicmaterial is saccharified with an enzyme composition in the presence of apolypeptide having cellobiohydrolase activity of the present invention.In one aspect, the fermenting of the cellulosic material produces afermentation product. In another aspect, the method further comprisesrecovering the fermentation product from the fermentation.

Definitions

Cellobiohydrolase: The term “cellobiohydrolase” means a1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91), which catalyzes thehydrolysis of 1,4-beta-D-glucosidic linkages in cellulose,cellooligosaccharides, or any beta-1,4-linked glucose containingpolymer, releasing cellobiose from the reducing or non-reducing ends ofthe chain (Teeri, 1997, Crystalline cellulose degradation: New insightinto the function of cellobiohydrolases, Trends in Biotechnology 15:160-167; Teeri et al., 1998, Trichoderma reesei cellobiohydrolases: whyso efficient on crystalline cellulose?, Biochem. Soc. Trans. 26:173-178). For purposes of the present invention, cellobiohydrolaseactivity is determined according to the procedures described by Lever etal., 1972, Anal. Biochem. 47: 273-279; van Tilbeurgh et al., 1982, FEBSLetters, 149: 152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters,187: 283-288; and Tomme et al., 1988, Eur. J. Biochem. 170: 575-581. Inthe present invention, the Lever et al. method can be employed to assesshydrolysis of cellulose in corn stover, while the methods of vanTilbeurgh et al. and Tomme et al. can be used to determine thecellobiohydrolase activity on a fluorescent disaccharide derivative,4-methylumbelliferyl-β-D-lactoside. Preferably the cellobiohydrolase ofthe invention is a family 7 glycosyl hydrolase (GH7). In the presentinvention, the assay described in the example section can be used tomeasure cellobiohydrolase activity.

The polypeptides of the present invention have at least 20%, e.g., atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, and at least 100% of the cellobiohydrolaseactivity of the mature polypeptide of SEQ ID NO: 2 or of SEQ ID NO: 4.

Cellulolytic Enzyme or Cellulase: The term “cellulolytic enzyme” or“cellulase” means one or more (several) enzymes that hydrolyze acellulosic material. Such enzymes include endoglucanase(s),cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof. Thetwo basic approaches for measuring cellulolytic activity include: (1)measuring the total cellulolytic activity, and (2) measuring theindividual cellulolytic activities (endoglucanases, cellobiohydrolases,and beta-glucosidases) as reviewed in Zhang et al., Outlook forcellulase improvement: Screening and selection strategies, 2006,Biotechnology Advances 24: 452-481. Total cellulolytic activity isusually measured using insoluble substrates, including Whatman No 1filter paper, microcrystalline cellulose, bacterial cellulose, algalcellulose, cotton, pretreated lignocellulose, etc. The most common totalcellulolytic activity assay is the filter paper assay using Whatman No 1filter paper as the substrate. The assay was established by theInternational Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987,Measurement of cellulase activities, Pure Appl. Chem. 59: 257-68).

For purposes of the present invention, cellulolytic enzyme activity isdetermined by measuring the increase in hydrolysis of a cellulosicmaterial by cellulolytic enzyme(s) under the following conditions: 1-20mg of cellulolytic enzyme protein/g of cellulose in PCS for 3-7 days at50° C. compared to a control hydrolysis without addition of cellulolyticenzyme protein. Typical conditions are 1 ml reactions, washed orunwashed PCS, 5% insoluble solids, 50 mM sodium acetate pH 5, 1 mMMnSO₄, 50° C., 72 hours, sugar analysis by AMINEX® HPX-87H column(Bio-Rad Laboratories, Inc., Hercules, Calif., USA).

Endoglucanase: The term “endoglucanase” means anendo-1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4),which catalyzes endohydrolysis of 1,4-beta-D-glycosidic linkages incellulose, cellulose derivatives (such as carboxymethyl cellulose andhydroxyethyl cellulose), lichenin, beta-1,4 bonds in mixed beta-1,3glucans such as cereal beta-D-glucans or xyloglucans, and other plantmaterial containing cellulosic components. Endoglucanase activity can bedetermined by measuring reduction in substrate viscosity or increase inreducing ends determined by a reducing sugar assay (Zhang et al., 2006,Biotechnology Advances 24: 452-481). For purposes of the presentinvention, endoglucanase activity is determined using carboxymethylcellulose (CMC) as substrate according to the procedure of Ghose, 1987,Pure and Appl. Chem. 59: 257-268, at pH 5, 40° C.

Beta-Glucosidase: The term “beta-glucosidase” means a beta-D-glucosideglucohydrolase (E.C. 3.2.1.21), which catalyzes the hydrolysis ofterminal non-reducing beta-D-glucose residues with the release ofbeta-D-glucose. For purposes of the present invention, beta-glucosidaseactivity is determined according to the basic procedure described byVenturi et al., 2002, Extracellular beta-D-glucosidase from Chaetomiumthermophilum var. coprophilum: production, purification and somebiochemical properties, J. Basic Microbiol. 42: 55-66. One unit ofbeta-glucosidase is defined as 1.0 μmole of p-nitrophenolate anionproduced per minute at 25° C., pH 4.8 from 1 mMp-nitrophenyl-beta-D-glucopyranoside as substrate in 50 mM sodiumcitrate containing 0.01% TWEEN® 20 (polyxyethylene sorbitanmonolaurate).

Polypeptide Having Cellulolytic Enhancing Activity: The term“polypeptide having cellulolytic enhancing activity” means a GH61polypeptide that catalyzes the enhancement of the hydrolysis of acellulosic material by enzyme having cellulolytic activity. For purposesof the present invention, cellulolytic enhancing activity is determinedby measuring the increase in reducing sugars or the increase of thetotal of cellobiose and glucose from the hydrolysis of a cellulosicmaterial by cellulolytic enzyme under the following conditions: 1-50 mgof total protein/g of cellulose in PCS, wherein total protein iscomprised of 50-99.5% w/w cellulolytic enzyme protein and 0.5-50% w/wprotein of a GH61 polypeptide having cellulolytic enhancing activity for1-7 days at 50° C. compared to a control hydrolysis with equal totalprotein loading without cellulolytic enhancing activity (1-50 mg ofcellulolytic protein/g of cellulose in PCS). In a preferred aspect, amixture of CELLUCLAST® 1.5 L (Novozymes A/S, Bagsævrd, Denmark) in thepresence of 2-3% of total protein weight Aspergillus oryzaebeta-glucosidase (recombinantly produced in Aspergillus oryzae accordingto WO 02/095014) or 2-3% of total protein weight Aspergillus fumigatusbeta-glucosidase (recombinantly produced in Aspergillus oryzae asdescribed in WO 2002/095014) of cellulase protein loading is used as thesource of the cellulolytic activity.

The GH61 polypeptides having cellulolytic enhancing activity enhance thehydrolysis of a cellulosic material catalyzed by enzyme havingcellulolytic activity by reducing the amount of cellulolytic enzymerequired to reach the same degree of hydrolysis preferably at least1.01-fold, more preferably at least 1.05-fold, more preferably at least1.10-fold, more preferably at least 1.25-fold, more preferably at least1.5-fold, more preferably at least 2-fold, more preferably at least3-fold, more preferably at least 4-fold, more preferably at least5-fold, even more preferably at least 10-fold, and most preferably atleast 20-fold.

Family 7 or Family 61 Glycoside Hydrolase: The term “Family GH7” or“GH7” or “Family 7 glycoside hydrolase” or “Family GH61” or “GH61” or“Family 61 glycoside hydrolase” means a polypeptide falling into theglycoside hydrolase Family 7 or Family 61, respectively, according toHenrissat, 1991, A classification of glycosyl hydrolases based onamino-acid sequence similarities, Biochem. J. 280: 309-316, andHenrissat and Bairoch, 1996, Updating the sequence-based classificationof glycosyl hydrolases, Biochem. J. 316: 695-696.

Hemicellulolytic Enzyme or Hemicellulase: The term “hemicellulolyticenzyme” or “hemicellulase” means one or more (several) enzymes thathydrolyze a hemicellulosic material. See, for example, Shallom andShoham, 2003, Microbial hemicellulases. Current Opinion In Microbiology6(3): 219-228). Hemicellulases are key components in the degradation ofplant biomass. Examples of hemicellulases include, but are not limitedto, an acetylmannan esterase, an acetyxylan esterase, an arabinanase, anarabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, agalactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, amannosidase, a xylanase, and a xylosidase. The substrates of theseenzymes, the hemicelluloses, are a heterogeneous group of branched andlinear polysaccharides that are bound via hydrogen bonds to thecellulose microfibrils in the plant cell wall, crosslinking them into arobust network. Hemicelluloses are also covalently attached to lignin,forming together with cellulose a highly complex structure. The variablestructure and organization of hemicelluloses require the concertedaction of many enzymes for its complete degradation. The catalyticmodules of hemicellulases are either glycoside hydrolases (GHs) thathydrolyze glycosidic bonds, or carbohydrate esterases (CEs), whichhydrolyze ester linkages of acetate or ferulic acid side groups. Thesecatalytic modules, based on homology of their primary sequence, can beassigned into GH and CE families marked by numbers. Some families, withoverall similar fold, can be further grouped into clans, markedalphabetically (e.g., GH-A). A most informative and updatedclassification of these and other carbohydrate active enzymes isavailable on the Carbohydrate-Active Enzymes (CAZy) database.Hemicellulolytic enzyme activities can be measured according to Ghoseand Bisaria, 1987, Pure & Appl. Chem. 59: 1739-1752.

Xylan Degrading Activity or Xylanolytic Activity: The term “xylandegrading activity” or “xylanolytic activity” means a biologicalactivity that hydrolyzes xylan-containing material. The two basicapproaches for measuring xylanolytic activity include: (1) measuring thetotal xylanolytic activity, and (2) measuring the individual xylanolyticactivities (e.g., endoxylanases, beta-xylosidases, arabinofuranosidases,alpha-glucuronidases, acetylxylan esterases, feruloyl esterases, andalpha-glucuronyl esterases). Recent progress in assays of xylanolyticenzymes was summarized in several publications including Biely andPuchard, Recent progress in the assays of xylanolytic enzymes, 2006,Journal of the Science of Food and Agriculture 86(11): 1636-1647;Spanikova and Biely, 2006, Glucuronoyl esterase—Novel carbohydrateesterase produced by Schizophyllum commune, FEBS Letters 580(19):4597-4601; Herrmann et al., 1997, The beta-D-xylosidase of Trichodermareesei is a multifunctional beta-D-xylan xylohydrolase, BiochemicalJournal 321: 375-381.

Total xylan degrading activity can be measured by determining thereducing sugars formed from various types of xylan, including, forexample, oat spelt, beechwood, and larchwood xylans, or by photometricdetermination of dyed xylan fragments released from various covalentlydyed xylans. The most common total xylanolytic activity assay is basedon production of reducing sugars from polymeric 4-O-methylglucuronoxylan as described in Bailey et al., 1992, Interlaboratorytesting of methods for assay of xylanase activity, Journal ofBiotechnology 23(3): 257-270. Xylanase activity can also be determinedwith 0.2% AZCL-arabinoxylan as substrate in 0.01% TRITON X-100(4-(1,1,3,3-tetramethylbutyl)phenyl-polythylene glycol) and 200 mMsodium phosphate buffer pH 6 at 37° C. One unit of xylanase activity isdefined as 1.0 μmole of azurine produced per minute at 37° C., pH 6 from0.2% AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6buffer.

For purposes of the present invention, xylan degrading activity isdetermined by measuring the increase in hydrolysis of birchwood xylan(Sigma Chemical Co., Inc., St. Louis, Mo., USA) by xylan-degradingenzyme(s) under the following typical conditions: 1 ml reactions, 5mg/ml substrate (total solids), 5 mg of xylanolytic protein/g ofsubstrate, 50 mM sodium acetate pH 5, 50° C., 24 hours, sugar analysisusing p-hydroxybenzoic acid hydrazide (PHBAH) assay as described byLever, 1972, A new reaction for colorimetric determination ofcarbohydrates, Anal. Biochem 47: 273-279.

Xylanase: The term “xylanase” means a 1,4-beta-D-xylan-xylohydrolase(E.C. 3.2.1.8) that catalyzes the endohydrolysis of 1,4-beta-D-xylosidiclinkages in xylans. For purposes of the present invention, xylanaseactivity is determined with 0.2% AZCL-arabinoxylan as substrate in 0.01%TRITON® X-100 and 200 mM sodium phosphate buffer pH 6 at 37° C. One unitof xylanase activity is defined as 1.0 μmole of azurine produced perminute at 37° C., pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200mM sodium phosphate pH 6 buffer.

Beta-Xylosidase: The term “beta-xylosidase” means a beta-D-xylosidexylohydrolase (E.C. 3.2.1.37) that catalyzes the exo-hydrolysis of shortbeta (1→4)-xylooligosaccharides, to remove successive D-xylose residuesfrom the non-reducing termini. For purposes of the present invention,one unit of beta-xylosidase is defined as 1.0 μmole of p-nitrophenolateanion produced per minute at 40° C., pH 5 from 1 mMp-nitrophenyl-beta-D-xyloside as substrate in 100 mM sodium citratecontaining 0.01% TWEEN® 20.

Acetylxylan Esterase: The term “acetylxylan esterase” means acarboxylesterase (EC 3.1.1.72) that catalyzes the hydrolysis of acetylgroups from polymeric xylan, acetylated xylose, acetylated glucose,alpha-napthyl acetate, and p-nitrophenyl acetate. For purposes of thepresent invention, acetylxylan esterase activity is determined using 0.5mM p-nitrophenylacetate as substrate in 50 mM sodium acetate pH 5.0containing 0.01% TWEEN™ 20. One unit of acetylxylan esterase is definedas the amount of enzyme capable of releasing 1 μmole of p-nitrophenolateanion per minute at pH 5, 25° C.

Feruloyl Esterase: The term “feruloyl esterase” means a4-hydroxy-3-methoxycinnamoyl-sugar hydrolase (EC 3.1.1.73) thatcatalyzes the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl (feruloyl)group from an esterified sugar, which is usually arabinose in “natural”substrates, to produce ferulate (4-hydroxy-3-methoxycinnamate). Feruloylesterase is also known as ferulic acid esterase, hydroxycinnamoylesterase, FAE-III, cinnamoyl ester hydrolase, FAEA, cinnAE, FAE-I, orFAE-II. For purposes of the present invention, feruloyl esteraseactivity is determined using 0.5 mM p-nitrophenylferulate as substratein 50 mM sodium acetate pH 5.0. One unit of feruloyl esterase equals theamount of enzyme capable of releasing 1 μmole of p-nitrophenolate anionper minute at pH 5, 25° C.

Alpha-Glucuronidase: The term “alpha-glucuronidase” means analpha-D-glucosiduronate glucuronohydrolase (EC 3.2.1.139) that catalyzesthe hydrolysis of an alpha-D-glucuronoside to D-glucuronate and analcohol. For purposes of the present invention, alpha-glucuronidaseactivity is determined according to de Vries, 1998, J. Bacteriol. 180:243-249. One unit of alpha-glucuronidase equals the amount of enzymecapable of releasing 1 μmole of glucuronic or 4-O-methylglucuronic acidper minute at pH 5, 40° C.

Alpha-L-Arabinofuranosidase: The term “alpha-L-arabinofuranosidase”means an alpha-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55)that catalyzes the hydrolysis of terminal non-reducingalpha-L-arabinofuranoside residues in alpha-L-arabinosides. The enzymeacts on alpha-L-arabinofuranosides, alpha-L-arabinans containing (1,3)-and/or (1,5)-linkages, arabinoxylans, and arabinogalactans.Alpha-L-arabinofuranosidase is also known as arabinosidase,alpha-arabinosidase, alpha-L-arabinosidase, alpha-arabinofuranosidase,polysaccharide alpha-L-arabinofuranosidase, alpha-L-arabinofuranosidehydrolase, L-arabinosidase, or alpha-L-arabinanase. For purposes of thepresent invention, alpha-L-arabinofuranosidase activity is determinedusing 5 mg of medium viscosity wheat arabinoxylan (MegazymeInternational Ireland, Ltd., Bray, Co. Wicklow, Ireland) per ml of 100mM sodium acetate pH 5 in a total volume of 200 μl for 30 minutes at 40°C. followed by arabinose analysis by AMINEX® HPX-87H columnchromatography (Bio-Rad Laboratories, Inc., Hercules, Calif., USA).

Cellulosic Material: The term “cellulosic material” means any materialcontaining cellulose. The predominant polysaccharide in the primary cellwall of biomass is cellulose, the second most abundant is hemicellulose,and the third is pectin. The secondary cell wall, produced after thecell has stopped growing, also contains polysaccharides and isstrengthened by polymeric lignin covalently cross-linked tohemicellulose. Cellulose is a homopolymer of anhydrocellobiose and thusa linear beta-(1-4)-D-glucan, while hemicelluloses include a variety ofcompounds, such as xylans, xyloglucans, arabinoxylans, and mannans incomplex branched structures with a spectrum of substituents. Althoughgenerally polymorphous, cellulose is found in plant tissue primarily asan insoluble crystalline matrix of parallel glucan chains.Hemicelluloses usually hydrogen bond to cellulose, as well as to otherhemicelluloses, which help stabilize the cell wall matrix.

Cellulose is generally found, for example, in the stems, leaves, hulls,husks, and cobs of plants or leaves, branches, and wood of trees. Thecellulosic material can be, but is not limited to, herbaceous material,agricultural residue, forestry residue, municipal solid waste, wastepaper, and pulp and paper mill residue (see, for example, Wiselogel etal., 1995, in Handbook on Bioethanol (Charles E. Wyman, editor), pp.105-118, Taylor & Francis, Washington D.C.; Wyman, 1994, BioresourceTechnology 50: 3-16; Lynd, 1990, Applied Biochemistry and Biotechnology24/25: 695-719; Mosier et al., 1999, Recent Progress in Bioconversion ofLignocellulosics, in Advances in Biochemical Engineering/Biotechnology,T. Scheper, managing editor, Volume 65, pp. 23-40, Springer-Verlag, NewYork). It is understood herein that the cellulose may be in the form oflignocellulose, a plant cell wall material containing lignin, cellulose,and hemicellulose in a mixed matrix. In a preferred aspect, thecellulosic material is lignocellulose, which comprises cellulose,hemicellulose, and lignin.

In one aspect, the cellulosic material is herbaceous material. Inanother aspect, the cellulosic material is agricultural residue. Inanother aspect, the cellulosic material is forestry residue. In anotheraspect, the cellulosic material is municipal solid waste. In anotheraspect, the cellulosic material is waste paper. In another aspect, thecellulosic material is pulp and paper mill residue.

In another aspect, the cellulosic material is corn stover. In anotheraspect, the cellulosic material is corn fiber. In another aspect, thecellulosic material is corn cob. In another aspect, the cellulosicmaterial is orange peel. In another aspect, the cellulosic material isrice straw. In another aspect, the cellulosic material is wheat straw.In another aspect, the cellulosic material is switch grass. In anotheraspect, the cellulosic material is miscanthus. In another aspect, thecellulosic material is bagasse.

In another aspect, the cellulosic material is microcrystallinecellulose. In another aspect, the cellulosic material is bacterialcellulose. In another aspect, the cellulosic material is algalcellulose. In another aspect, the cellulosic material is cotton linter.In another aspect, the cellulosic material is amorphous phosphoric-acidtreated cellulose. In another aspect, the cellulosic material is filterpaper.

The cellulosic material may be used as is or may be subjected topretreatment, using conventional methods known in the art, as describedherein. In a preferred aspect, the cellulosic material is pretreated.

Pretreated Corn Stover: The term “PCS” or “Pretreated Corn Stover” meansa cellulosic material derived from corn stover by treatment with heatand diluted sulfuric acid.

Very Low Stringency Conditions: the term “very low stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 45° C.

Low Stringency Conditions: The term “low stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 25% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at50° C.

Medium Stringency Conditions: The term “medium stringency conditions”means for probes of at least 100 nucleotides in length, prehybridizationand hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/mlsheared and denatured salmon sperm DNA, and 35% formamide, followingstandard Southern blotting procedures for 12 to 24 hours. The carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS at 55° C.

Medium-High Stringency Conditions: The term “medium-high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and either 35%formamide, following standard Southern blotting procedures for 12 to 24hours. The carrier material is finally washed three times each for 15minutes using 2×SSC, 0.2% SDS at 60° C.

High Stringency Conditions: The term “high stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 50% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at65° C.

Very High Stringency Conditions: The term “very high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 70° C.

Isolated: The term “isolated” means a substance in a form or environmentthat does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., multiple copiesof a gene encoding the substance; use of a stronger promoter than thepromoter naturally associated with the gene encoding the substance). Anisolated substance may be present in a fermentation broth sample.

Mature Polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. In one aspect, the maturepolypeptide is amino acids 19 to 455 of SEQ ID NO: 2 based on theSignalP program (Nielsen et al., 1997, Protein Engineering 10: 1-6) thatpredicts amino acids 1 to 18 of SEQ ID NO: 2 are a signal peptide. It isknown in the art that a host cell may produce a mixture of two of moredifferent mature polypeptides (i.e., with a different C-terminal and/orN-terminal amino acid) expressed by the same polynucleotide. In anotheraspect the mature polypeptide is amino acids 26 to 537 of SEQ ID NO: 4based on the SignalP program (Nielsen et al., 1997, Protein Engineering10: 1-6) that predicts amino acids 1 to 25 of SEQ ID NO: 4 are a signalpeptide. It is known in the art that a host cell may produce a mixtureof two of more different mature polypeptides (i.e., with a differentC-terminal and/or N-terminal amino acid) expressed by the samepolynucleotide

Mature Polypeptide Coding Sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving cellobiohydrolase activity. In one aspect, the mature polypeptidecoding sequence is nucleotides 55 to 603, 668 to 1235, 1311 to 1507 ofSEQ ID NO: 1 based on the SignalP program (Nielsen et al., 1997, supra)that predicts nucleotides 1 to 54 of SEQ ID NO: 1 encode a signalpeptide. In another aspect, the mature polypeptide coding sequence isnucleotides 76 to 1614 of SEQ ID NO: 3 based on the SignalP program(Nielsen et al., 1997, supra) that predicts nucleotides 1 to 75 of SEQID NO: 3 encode a signal peptide.

Catalytic Domain: The term “catalytic domain” means the portion of anenzyme containing the catalytic machinery of the enzyme.

Cellulose Binding Domain: The term “cellulose binding domain” means theportion of an enzyme that mediates binding of the enzyme to amorphousregions of a cellulose substrate. The cellulose binding domain (CBD) isfound either at the N-terminal or at the C-terminal extremity of anenzyme. A CBD is also referred to as a cellulose binding module or CBM.In one embodiment the CBM is amino acids 502 to 537 of SEQ ID NO: 4. TheCBM is separated from the catalytic domain by a linker sequence. Thelinker is in one embodiment amino acids 452 to 495 of SEQ ID NO: 4.

Sequence Identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the degree of sequence identitybetween two amino acid sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.48: 443-453) as implemented in the Needle program of the EMBOSS package(EMBOSS: The European Molecular Biology Open Software Suite, Rice etal., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 orlater. The optional parameters used are gap open penalty of 10, gapextension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix. The output of Needle labeled “longest identity”(obtained using the -nobrief option) is used as the percent identity andis calculated as follows:(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the degree of sequence identitybetween two deoxyribonucleotide sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,supra), preferably version 5.0.0 or later. The optional parameters usedare gap open penalty of 10, gap extension penalty of 0.5, and theEDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The outputof Needle labeled “longest identity” (obtained using the -nobriefoption) is used as the percent identity and is calculated as follows:(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Fragment: The term “fragment” means a polypeptide having one or more(several) amino acids deleted from the amino and/or carboxyl terminus ofa mature polypeptide; wherein the fragment has cellobiohydrolaseactivity. In one aspect, a fragment contains at least 390 amino acidresidues, e.g., at least 410 amino acid residues or at least 430 aminoacid residues.

Subsequence: The term “subsequence” means a polynucleotide having one ormore (several) nucleotides deleted from the 5′ and/or 3′ end of a maturepolypeptide coding sequence; wherein the subsequence encodes a fragmenthaving cellobiohydrolase activity. In one aspect, a subsequence containsat least 1170 nucleotides, e.g., at least 1230 nucleotides or at least1290 nucleotides

Allelic Variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Coding Sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a polypeptide. Theboundaries of the coding sequence are generally determined by an openreading frame, which usually begins with the ATG start codon oralternative start codons such as GTG and TTG and ends with a stop codonsuch as TAA, TAG, and TGA. The coding sequence may be a DNA, cDNA,synthetic, or recombinant polynucleotide.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic cell. cDNA lacks intron sequences that may be presentin the corresponding genomic DNA. The initial, primary RNA transcript isa precursor to mRNA that is processed through a series of steps,including splicing, before appearing as mature spliced mRNA.

Nucleic Acid Construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic. The term nucleic acid construct issynonymous with the term “expression cassette” when the nucleic acidconstruct contains the control sequences required for expression of acoding sequence of the present invention.

Control Sequences: The term “control sequences” means all componentsnecessary for the expression of a polynucleotide encoding a polypeptideof the present invention. Each control sequence may be native or foreignto the polynucleotide encoding the polypeptide or native or foreign toeach other. Such control sequences include, but are not limited to, aleader, polyadenylation sequence, propeptide sequence, promoter, signalpeptide sequence, and transcription terminator. At a minimum, thecontrol sequences include a promoter, and transcriptional andtranslational stop signals. The control sequences may be provided withlinkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe polynucleotide encoding a polypeptide.

Operably Linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs the expression of the coding sequence.

Expression: The term “expression” includes any step involved in theproduction of the polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression Vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding apolypeptide and is operably linked to additional nucleotides thatprovide for its expression.

Host Cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, and the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Variant: The term “variant” means a polypeptide having cellobiohydrolaseactivity comprising an alteration, i.e., a substitution, insertion,and/or deletion of one or more (several) amino acid residues at one ormore (several) positions. A substitution means a replacement of an aminoacid occupying a position with a different amino acid; a deletion meansremoval of an amino acid occupying a position; and an insertion meansadding one or more (several) amino acids, e.g., 1-5 amino acids,adjacent to an amino acid occupying a position.

DETAILED DESCRIPTION OF THE INVENTION

Polypeptides Having Cellobiohydrolase Activity

The present invention relates to isolated polypeptides havingcellobiohydrolase activity selected from the group consisting of:

(a) a polypeptide having at least 90% sequence identity to the maturepolypeptide of SEQ ID NO: 2 or a polypeptide having at least 80%sequence identity to the mature polypeptide of SEQ ID NO: 4;

(b) a polypeptide encoded by a polynucleotide having at least 90%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 or the cDNA sequence thereof or a polypeptide encoded by apolynucleotide having at least 80% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 3 or the cDNA sequencethereof;

(c) a variant comprising a substitution, deletion, and/or insertion ofone or more (several) amino acids of the mature polypeptide of SEQ IDNO: 2 or of SEQ ID NO: 4; and

(d) a fragment of the polypeptide of (a), (b), or (c) that hascellobiohydrolase activity.

The present invention relates to isolated polypeptides having a sequenceidentity to the mature polypeptide of SEQ ID NO: 2 of at least 90%, e.g.at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%, which have cellobiohydrolase activity. In oneaspect, the polypeptides differ by no more than ten amino acids, e.g.,by five amino acids, by four amino acids, by three amino acids, by twoamino acids, and by one amino acid from the mature polypeptide of SEQ IDNO: 2.

A polypeptide of the present invention preferably comprises or consistsof the amino acid sequence of SEQ ID NO: 2 or an allelic variantthereof; or is a fragment thereof having cellobiohydrolase activity. Inanother aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 2. In another preferred aspect, thepolypeptide comprises or consists of amino acids 19 to 469 of SEQ ID NO:2.

The present invention relates to isolated polypeptides having a sequenceidentity to the mature polypeptide of SEQ ID NO: 4 of at least 80%, e.g.at least 85%, at least 87%, at least 90%, at least 92%, at least 95%,e.g., at least 96%, at least 97%, at least 98%, at least 99%, or 100%,which have cellobiohydrolase activity. In one aspect, the polypeptidesdiffer by no more than ten amino acids, e.g., by five amino acids, byfour amino acids, by three amino acids, by two amino acids, and by oneamino acid from the mature polypeptide of SEQ ID NO: 4.

A polypeptide of the present invention preferably comprises or consistsof the amino acid sequence of SEQ ID NO: 4 or an allelic variantthereof; or is a fragment thereof having cellobiohydrolase activity. Inanother aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 4. In another preferred aspect, thepolypeptide comprises or consists of amino acids 26 to 537 of SEQ ID NO:4. In another aspect the polypeptide comprises or consist of amino acids26 to 465 of SEQ ID NO: 4. This fragment comprises the catalytic domain.

The present invention also relates to isolated polypeptides havingcellobiohydrolase activity that are encoded by polynucleotides thathybridize under very low stringency conditions, low stringencyconditions, medium stringency conditions, medium-high stringencyconditions, high stringency conditions, or very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the cDNA sequence contained in the mature polypeptide codingsequence of SEQ ID NO: 1, or (iii) the full-length complementary strandof (i) or (ii) (J. Sambrook, E.F. Fritsch, and T. Maniatis, 1989,Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor,N.Y.).

The present invention also relates to isolated polypeptides havingcellobiohydrolase activity that are encoded by polynucleotides thathybridize under very low stringency conditions, low stringencyconditions, medium stringency conditions, medium-high stringencyconditions, high stringency conditions, or very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:3, (ii) the genomic DNA sequence of SEQ ID NO: 3, or (iii) thefull-length complementary strand of (i) or (ii) (J. Sambrook, E.F.Fritsch, and T. Maniatis, 1989, Molecular Cloning, A Laboratory Manual,2d edition, Cold Spring Harbor, N.Y.).

The polynucleotide of SEQ ID NO: 1 or SEQ ID NO: 3, or a subsequencethereof, as well as the amino acid sequence of SEQ ID NO: 2 or SEQ IDNO: 4, or a fragment thereof, may be used to design nucleic acid probesto identify and clone DNA encoding polypeptides having cellobiohydrolaseactivity from strains of different genera or species according tomethods well known in the art. In particular, such probes can be usedfor hybridization with the genomic DNA or cDNA of the genus or speciesof interest, following standard Southern blotting procedures, in orderto identify and isolate the corresponding gene therein. Such probes canbe considerably shorter than the entire sequence, but should be at least14, e.g., at least 25, at least 35, or at least 70 nucleotides inlength. Preferably, the nucleic acid probe is at least 100 nucleotidesin length, e.g., at least 200 nucleotides, at least 300 nucleotides, atleast 400 nucleotides, at least 500 nucleotides, at least 600nucleotides, at least 700 nucleotides, at least 800 nucleotides, or atleast 900 nucleotides in length. Both DNA and RNA probes can be used.The probes are typically labeled for detecting the corresponding gene(for example, with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes areencompassed by the present invention.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a polypeptide having cellobiohydrolase activity. Genomic orother DNA from such other strains may be separated by agarose orpolyacrylamide gel electrophoresis, or other separation techniques. DNAfrom the libraries or the separated DNA may be transferred to andimmobilized on nitrocellulose or other suitable carrier material. Inorder to identify a clone or DNA that is homologous with SEQ ID NO: 1 ora subsequence thereof, the carrier material is preferably used in aSouthern blot.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labeled nucleic acid probe correspondingto SEQ ID NO: 1 or SEQ ID NO: 3; the mature polypeptide coding sequenceof SEQ ID NO: 1 or SEQ ID NO: 3; the cDNA sequence contained in themature polypeptide coding sequence of SEQ ID NO: 1 or the genomic DNAsequence of SEQ ID NO: 3; its full-length complementary strand; or asubsequence thereof; under very low to very high stringency conditions.Molecules to which the nucleic acid probe hybridizes under theseconditions can be detected using, for example, X-ray film.

In one aspect, the nucleic acid probe is the mature polypeptide codingsequence of SEQ ID NO: 1 or the cDNA sequence thereof. In anotheraspect, the nucleic acid probe is nucleotides 55 to 1507 of SEQ ID NO: 1or the cDNA sequence thereof. In another aspect, the nucleic acid probeis a polynucleotide that encodes the polypeptide of SEQ ID NO: 2 or themature polypeptide thereof; or a fragment thereof. In another preferredaspect, the nucleic acid probe is SEQ ID NO: 1 or the cDNA sequencethereof.

In one aspect, the nucleic acid probe is the mature polypeptide codingsequence of SEQ ID NO: 3 or the genomic DNA sequence thereof. In anotheraspect, the nucleic acid probe is nucleotides 76 to 1614 of SEQ ID NO: 3or the genomic sequence thereof. In another aspect, the nucleic acidprobe is a polynucleotide that encodes the polypeptide of SEQ ID NO: 4or the mature polypeptide thereof; or a fragment thereof. In anotherpreferred aspect, the nucleic acid probe is SEQ ID NO: 3 or the genomicsequence thereof.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and either 25% formamide for very lowand low stringencies, 35% formamide for medium and medium-highstringencies, or 50% formamide for high and very high stringencies,following standard Southern blotting procedures for 12 to 24 hoursoptimally. The carrier material is finally washed three times each for15 minutes using 2×SSC, 0.2% SDS at 45° C. (very low stringency), at 50°C. (low stringency), at 55° C. (medium stringency), at 60° C.(medium-high stringency), at 65° C. (high stringency), and at 70° C.(very high stringency).

For short probes of about 15 nucleotides to about 70 nucleotides inlength, stringency conditions are defined as prehybridization andhybridization at about 5° C. to about 10° C. below the calculated T_(m)using the calculation according to Bolton and McCarthy (1962, Proc.Natl. Acad. Sci. USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6mM EDTA, 0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA perml following standard Southern blotting procedures for 12 to 24 hoursoptimally. The carrier material is finally washed once in 6×SCC plus0.1% SDS for 15 minutes and twice each for 15 minutes using 6×SSC at 5°C. to 10° C. below the calculated T_(m).

The present invention also relates to isolated polypeptides havingcellobiohydrolase activity encoded by polynucleotides having a sequenceidentity to the mature polypeptide coding sequence of SEQ ID NO: 1 orthe cDNA sequence thereof of at least 90%, e.g. at least 92%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%.

The present invention also relates to isolated polypeptides havingcellobiohydrolase activity encoded by polynucleotides having a sequenceidentity to the mature polypeptide coding sequence of SEQ ID NO: 3 orthe genomic sequence thereof of at least 80%, e.g. at least 85%, atleast 87%, at least 90%, at least 92%, at least 95%, e.g., at least 96%,at least 97%, at least 98%, at least 99%, or 100%.

The present invention also relates to variants comprising asubstitution, deletion, and/or insertion of one or more (or several)amino acids of the mature polypeptide of SEQ ID NO: 2 or of SEQ ID NO:4, or a homologous sequences thereof. Preferably, amino acid changes areof a minor nature, that is conservative amino acid substitutions orinsertions that do not significantly affect the folding and/or activityof the protein; small deletions, typically of one to about 30 aminoacids; small amino- or carboxyl-terminal extensions, such as anamino-terminal methionine residue; a small linker peptide of up to about20-25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in a parent polypeptide can be identifiedaccording to procedures known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989,Science 244: 1081-1085). In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for cellobiohydrolase activity toidentify amino acid residues that are critical to the activity of themolecule. See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708.The active site of the enzyme or other biological interaction can alsobe determined by physical analysis of structure, as determined by suchtechniques as nuclear magnetic resonance, crystallography, electrondiffraction, or photoaffinity labeling, in conjunction with mutation ofputative contact site amino acids. See, for example, de Vos et al.,1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identities ofessential amino acids can also be inferred from analysis of identitieswith polypeptides that are related to the parent polypeptide.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

The total number of amino acid substitutions, deletions and/orinsertions of the mature polypeptides of SEQ ID NO: 2 or SEQ ID NO: 4are in one embodiment not more than 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8 or9.

The polypeptide may be hybrid polypeptide in which a portion of onepolypeptide is fused at the N-terminus or the C-terminus of a portion ofanother polypeptide.

The polypeptide may be a fused polypeptide or cleavable fusionpolypeptide in which another polypeptide is fused at the N-terminus orthe C-terminus of the polypeptide of the present invention. A fusedpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fused polypeptide is under control of thesame promoter(s) and terminator. Fusion proteins may also be constructedusing intein technology in which fusions are createdpost-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawsonet al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

Sources of Polypeptides Having Cellobiohydrolase Activity

A polypeptide having cellobiohydrolase activity of the present inventionmay be obtained from microorganisms of any genus. For purposes of thepresent invention, the term “obtained from” as used herein in connectionwith a given source shall mean that the polypeptide encoded by apolynucleotide is produced by the source or by a strain in which thepolynucleotide from the source has been inserted. In one aspect, thepolypeptide obtained from a given source is secreted extracellularly.

The polypeptide may be a Talaromyces polypeptide.

In another aspect, the polypeptide is a Talaromyces byssochlamydoidespolypeptide. In another aspect, the polypeptide is a Talaromycesbyssochlamydoides CBS413.71 polypeptide.

It will be understood that for the aforementioned species the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The polypeptide may be identified and obtained from other sourcesincluding microorganisms isolated from nature (e.g., soil, composts,water, etc.) using the above-mentioned probes. Techniques for isolatingmicroorganisms from natural habitats are well known in the art. Thepolynucleotide encoding the polypeptide may then be obtained bysimilarly screening a genomic DNA or cDNA library of anothermicroorganism or mixed DNA sample. Once a polynucleotide encoding apolypeptide has been detected with the probe(s), the polynucleotide canbe isolated or cloned by utilizing techniques that are well known tothose of ordinary skill in the art (see, e.g., Sambrook et al., 1989,supra).

Catalytic Domains

The present invention also relates to isolated polypeptides comprising acatalytic domain selected from the group consisting of:

(a) a catalytic domain having at least 90% sequence identity to thecatalytic domain of SEQ ID NO: 2 or a catalytic domain having at least80% sequence identity to the catalytic domain of SEQ ID NO: 4;

(b) a catalytic domain encoded by a polynucleotide having at least 90%sequence identity to the catalytic domain coding sequence of SEQ ID NO:1 or a catalytic domain encoded by a polynucleotide having at least 80%sequence identity to the catalytic domain coding sequence of SEQ ID NO:3;

(c) a variant of a catalytic domain comprising a substitution, deletion,and/or insertion of one or more (several) amino acids of the catalyticdomain of SEQ ID NO: 2 or of SEQ ID NO: 4; and

(d) a fragment of a catalytic domain of (a), (b), or (c), which hascellobiohydrolase activity.

The catalytic domain preferably has a degree of sequence identity to thecatalytic domain of SEQ ID NO: 2 of at least 90%, e.g. at least 92%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100%. In an aspect, the catalytic domain comprises an amino acidsequence that differs by ten amino acids, e.g., by five amino acids, byfour amino acids, by three amino acids, by two amino acids, and by oneamino acid from the catalytic domain of SEQ ID NO: 2.

The catalytic domain preferably comprises or consists of the catalyticdomain of SEQ ID NO: 2 or an allelic variant thereof; or is a fragmentthereof having cellobiohydrolase activity. In another aspect, thecatalytic domain comprises or consists of the catalytic domain of SEQ IDNO: 2. In another preferred aspect, the catalytic domain comprises orconsists of amino acids 19 to 455 of SEQ ID NO: 2.

The catalytic domain preferably has a degree of sequence identity to thecatalytic domain of SEQ ID NO: 4 of at least 80%, e.g. at least 85%, atleast 87%, at least 90%, at least 92%, at least 95%, e.g., at least 96%,at least 97%, at least 98%, at least 99%, or 100%. In an aspect, thecatalytic domain comprises an amino acid sequence that differs by tenamino acids, e.g., by five amino acids, by four amino acids, by threeamino acids, by two amino acids, and by one amino acid from thecatalytic domain of SEQ ID NO: 4.

The catalytic domain preferably comprises or consists of the catalyticdomain of SEQ ID NO: 4 or an allelic variant thereof; or is a fragmentthereof having cellobiohydrolase activity. In another aspect, thecatalytic domain comprises or consists of the catalytic domain of SEQ IDNO: 4. In another preferred aspect, the catalytic domain comprises orconsists of amino acids 26 to 465 of SEQ ID NO: 4.

In an embodiment, the catalytic domain may be encoded by apolynucleotide that hybridizes under very low stringency conditions, lowstringency conditions, medium stringency conditions, medium-highstringency conditions, high stringency conditions, and very highstringency conditions (as defined above) with (i) the catalytic domaincoding sequence of SEQ ID NO: 1, (ii) the cDNA sequence contained in thecatalytic domain coding sequence of SEQ ID NO: 1, or (iii) thefull-length complementary strand of (i) or (ii) (J. Sambrook et al.,1989, supra).

The catalytic domain may be encoded by a polynucleotide having a degreeof sequence identity to the catalytic domain coding sequence of SEQ IDNO: 1 of at least 90%, e.g. at least 92%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%, which encode apolypeptide having cellobiohydrolase activity.

In one aspect, the polynucleotide encoding the catalytic domaincomprises or consists of nucleotides 55 to 1507 of SEQ ID NO: 1 or thecDNA sequence thereof. In particular the polynucleotide encoding thecatalytic domain comprises or consists of nucleotides 55 to 603, 668 to1235, 1311 to 1507 of SEQ ID NO: 1.

In another embodiment, the catalytic domain may be encoded by apolynucleotide that hybridizes under very low stringency conditions, lowstringency conditions, medium stringency conditions, medium-highstringency conditions, high stringency conditions, and very highstringency conditions (as defined above) with (i) the catalytic domaincoding sequence of SEQ ID NO: 3, (ii) the genomic DNA sequence of SEQ IDNO: 3, or (iii) the full-length complementary strand of (i) or (ii) (J.Sambrook et al., 1989, supra).

The catalytic domain may be encoded by a polynucleotide having a degreeof sequence identity to the catalytic domain coding sequence of SEQ IDNO: 3 of at least 80%, e.g. at least 85%, at least 87%, at least 90%, atleast 92%, at least 95%, e.g., at least 96%, at least 97%, at least 98%,at least 99%, or 100%, which encode a polypeptide havingcellobiohydrolase activity.

In one aspect, the polynucleotide encoding the catalytic domaincomprises or consists of nucleotides 76 to 1395 of SEQ ID NO: 3 or thecDNA sequence thereof.

Polynucleotides

The present invention also relates to isolated polynucleotides encodinga polypeptide of the present invention.

The techniques used to isolate or clone a polynucleotide encoding apolypeptide are known in the art and include isolation from genomic DNA,preparation from cDNA, or a combination thereof. The cloning of thepolynucleotides from such genomic DNA can be effected, e.g., by usingthe well known polymerase chain reaction (PCR) or antibody screening ofexpression libraries to detect cloned DNA fragments with sharedstructural features. See, e.g., Innis et al., 1990, PCR: A Guide toMethods and Application, Academic Press, New York. Other nucleic acidamplification procedures such as ligase chain reaction (LCR), ligationactivated transcription (LAT) and polynucleotide-based amplification(NASBA) may be used. The polynucleotides may be cloned from a strain ofPenicillium, or a related organism and thus, for example, may be anallelic or species variant of the polypeptide encoding region of thepolynucleotide.

The present invention also relates to isolated polynucleotidescomprising or consisting of polynucleotides having a degree of sequenceidentity to the mature polypeptide coding sequence of SEQ ID NO: 1 orthe cDNA sequence thereof of at least 90%, e.g. at least 92%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%,which encode a polypeptide having cellobiohydrolase activity.

The present invention further relates to isolated polynucleotidescomprising or consisting of polynucleotides having a degree of sequenceidentity to the mature polypeptide coding sequence of SEQ ID NO: 3 orthe genomic DNA sequence thereof of at least 80%, e.g. at least 85%, atleast 87%, at least 90%, at least 92%, at least 95%, e.g., at least 96%,at least 97%, at least 98%, at least 99%, or 100%, which encode apolypeptide having cellobiohydrolase activity.

Modification of a polynucleotide encoding a polypeptide of the presentinvention may be necessary for the synthesis of polypeptidessubstantially similar to the polypeptide. The term “substantiallysimilar” to the polypeptide refers to non-naturally occurring forms ofthe polypeptide. These polypeptides may differ in some engineered wayfrom the polypeptide isolated from its native source, e.g., variantsthat differ in specific activity, thermostability, pH optimum, or thelike. The variant may be constructed on the basis of the polynucleotidepresented as the mature polypeptide coding sequence of SEQ ID NO: 1 orthe cDNA sequence thereof, e.g., a subsequence thereof, and/or byintroduction of nucleotide substitutions that do not result in a changein the amino acid sequence of the polypeptide, but which correspond tothe codon usage of the host organism intended for production of theenzyme, or by introduction of nucleotide substitutions that may giverise to a different amino acid sequence. For a general description ofnucleotide substitution, see, e.g., Ford et al., 1991, ProteinExpression and Purification 2: 95-107.

The present invention also relates to isolated polynucleotides encodingpolypeptides of the present invention, which hybridize under very lowstringency conditions, low stringency conditions, medium stringencyconditions, medium-high stringency conditions, high stringencyconditions, or very high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 1, (ii) the cDNA sequencecontained in the mature polypeptide coding sequence of SEQ ID NO: 1, or(iii) the full-length complementary strand of (i) or (ii); or allelicvariants and subsequences thereof (Sambrook et al., 1989, supra), asdefined herein.

In one aspect, the polynucleotide comprises or consists of SEQ ID NO: 1,the mature polypeptide coding sequence of SEQ ID NO: 1, or a subsequenceof SEQ ID NO: 1 that encodes a fragment of SEQ ID NO: 2 havingcellobiohydrolase activity, such as the polynucleotide of nucleotides 55to 1507 of SEQ ID NO: 1, as well as the cDNA sequences thereof.

In another aspect, the polynucleotide comprises or consists of thecatalytic domain coding sequence of SEQ ID NO: 1, such as thepolynucleotide of nucleotides 55 to 603, 668 to 1235, 1311 to 1507 ofSEQ ID NO: 1.

The present invention also relates to isolated polynucleotides encodingpolypeptides of the present invention, which hybridize under very lowstringency conditions, low stringency conditions, medium stringencyconditions, medium-high stringency conditions, high stringencyconditions, or very high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 3, (ii) the genomic DNAsequence contained in the mature polypeptide coding sequence of SEQ IDNO: 3, or (iii) the full-length complementary strand of (i) or (ii); orallelic variants and subsequences thereof (Sambrook et al., 1989,supra), as defined herein.

In one aspect, the polynucleotide comprises or consists of SEQ ID NO: 3,the mature polypeptide coding sequence of SEQ ID NO: 3, or a subsequenceof SEQ ID NO: 3 that encodes a fragment of SEQ ID NO: 4 havingcellobiohydrolase activity, such as the polynucleotide of nucleotides 76to 1614 of SEQ ID NO: 3, as well as the genomic DNA sequences thereof.

In another aspect, the polynucleotide comprises or consists of thecatalytic domain coding sequence of SEQ ID NO: 3, such as thepolynucleotide of nucleotides 76 to 1395 of SEQ ID NO: 3, as well as thegenomic DNA sequences thereof.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide of the present invention operably linked to one or more(several) control sequences that direct the expression of the codingsequence in a suitable host cell under conditions compatible with thecontrol sequences.

A polynucleotide may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the polynucleotide priorto its insertion into a vector may be desirable or necessary dependingon the expression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter sequence, a polynucleotide thatis recognized by a host cell for expression of a polynucleotide encodinga polypeptide of the present invention. The promoter sequence containstranscriptional control sequences that mediate the expression of thepolypeptide. The promoter may be any polynucleotide that showstranscriptional activity in the host cell of choice including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xylA and xylB genes, E. colilac operon, Streptomyces coelicolor agarase gene (dagA), and prokaryoticbeta-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci.USA 75: 3727-3731), as well as the tac promoter (DeBoer et al., 1983,Proc. Natl. Acad. Sci. USA 80: 21-25). Further promoters are describedin “Useful proteins from recombinant bacteria” in Gilbert et al., 1980,Scientific American, 242: 74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a modified promoter from a gene encoding a neutralalpha-amylase in Aspergilli in which the untranslated leader has beenreplaced by an untranslated leader from a gene encoding triose phosphateisomerase in Aspergilli; non-limiting examples include modifiedpromoters from the gene encoding neutral alpha-amylase in Aspergillusniger in which the untranslated leader has been replaced by anuntranslated leader from the gene encoding triose phosphate isomerase inAspergillus nidulans or Aspergillus oryzae); and mutant, truncated, andhybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, which is recognized by a host cell to terminate transcription.The terminator sequence is operably linked to the 3′-terminus of thepolynucleotide encoding the polypeptide. Any terminator that isfunctional in the host cell of choice may be used in the presentinvention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans anthranilate synthase,Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase,Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-likeprotease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, whentranscribed is a nontranslated region of an mRNA that is important fortranslation by the host cell. The leader sequence is operably linked tothe 5′-terminus of the polynucleotide encoding the polypeptide. Anyleader sequence that is functional in the host cell of choice may beused.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the polynucleotide and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell of choice may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a polypeptide anddirects the polypeptide into the cell's secretory pathway. The 5′-end ofthe coding sequence of the polynucleotide may inherently contain asignal peptide coding sequence naturally linked in translation readingframe with the segment of the coding sequence that encodes thepolypeptide. Alternatively, the 5′-end of the coding sequence maycontain a signal peptide coding sequence that is foreign to the codingsequence. The foreign signal peptide coding sequence may be requiredwhere the coding sequence does not naturally contain a signal peptidecoding sequence. Alternatively, the foreign signal peptide codingsequence may simply replace the natural signal peptide coding sequencein order to enhance secretion of the polypeptide. However, any signalpeptide coding sequence that directs the expressed polypeptide into thesecretory pathway of a host cell of choice may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a polypeptide. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present at theN-terminus of a polypeptide, the propeptide sequence is positioned nextto the N-terminus of a polypeptide and the signal peptide sequence ispositioned next to the N-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those that causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems. In yeast, the ADH2 system or GAL1 systemmay be used. In filamentous fungi, the Aspergillus niger glucoamylasepromoter, Aspergillus oryzae TAKA alpha-amylase promoter, andAspergillus oryzae glucoamylase promoter may be used. Other examples ofregulatory sequences are those that allow for gene amplification. Ineukaryotic systems, these regulatory sequences include the dihydrofolatereductase gene that is amplified in the presence of methotrexate, andthe metallothionein genes that are amplified with heavy metals. In thesecases, the polynucleotide encoding the polypeptide would be operablylinked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleotideand control sequences may be joined together to produce a recombinantexpression vector that may include one or more (several) convenientrestriction sites to allow for insertion or substitution of thepolynucleotide encoding the polypeptide at such sites. Alternatively,the polynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the sequence into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more (several) selectable markersthat permit easy selection of transformed, transfected, transduced, orthe like cells. A selectable marker is a gene the product of whichprovides for biocide or viral resistance, resistance to heavy metals,prototrophy to auxotrophs, and the like.

Examples of bacterial selectable markers are the dal genes from Bacillussubtilis or Bacillus licheniformis, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, ortetracycline resistance. Suitable markers for yeast host cells are ADE2,HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in afilamentous fungal host cell include, but are not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Preferred for use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMß1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a polypeptide. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide of the present invention operably linked to one or more(several) control sequences that direct the production of a polypeptideof the present invention. A construct or vector comprising apolynucleotide is introduced into a host cell so that the construct orvector is maintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thepolypeptide and its source.

The host cell may be any cell useful in the recombinant production of apolypeptide of the present invention, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any gram-positive or gram-negativebacterium. Gram-positive bacteria include, but not limited to, Bacillus,Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus,Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces.Gram-negative bacteria include, but not limited to, Campylobacter, E.coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter,Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell including, butnot limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may, for instance, beeffected by protoplast transformation (see, e.g., Chang and Cohen, 1979,Mol. Gen. Genet. 168: 111-115), by using competent cells (see, e.g.,Young and Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), by electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or byconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may, forinstance, be effected by protoplast transformation (see, e.g., Hanahan,1983, J. Mol. Biol. 166: 557-580) or electroporation (see, e.g., Doweret al., 1988, Nucleic Acids Res. 16: 6127-6145). The introduction of DNAinto a Streptomyces cell may, for instance, be effected by protoplasttransformation and electroporation (see, e.g., Gong et al., 2004, FoliaMicrobiol. (Praha) 49: 399-405), by conjugation (see, e.g., Mazodier etal., 1989, J. Bacteriol. 171: 3583-3585), or by transduction (see, e.g.,Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98: 6289-6294). Theintroduction of DNA into a Pseudomonas cell may, for instance, beeffected by electroporation (see, e.g., Choi et al., 2006, J. Microbiol.Methods 64: 391-397) or by conjugation (see, e.g., Pinedo and Smets,2005, Appl. Environ. Microbiol. 71: 51-57). The introduction of DNA intoa Streptococcus cell may, for instance, be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), by protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), by electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or by conjugation(see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, anymethod known in the art for introducing DNA into a host cell can beused.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (asdefined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary ofThe Fungi, 8th edition, 1995, CAB International, University Press,Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al.,1995, supra, page 171) and all mitosporic fungi (Hawksworth et al.,1995, supra).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, F.A., Passmore, S. M., andDavenport, R.R., eds, Soc. App. Bacteriol. Symposium Series No. 9,1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phiebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsuiphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phiebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a polypeptideof the present invention, comprising: (a) cultivating a cell, which inits wild-type form produces the polypeptide, under conditions conducivefor production of the polypeptide; and (b) recovering the polypeptide.In a preferred aspect, the cell is of the genus Talaromyces. In a morepreferred aspect, the cell is Talaromyces byssochlamydoides. In a mostpreferred aspect, the cell is Talaromyces byssochlamydoides strainCBS413.71.

The present invention also relates to methods of producing a polypeptideof the present invention, comprising: (a) cultivating a recombinant hostcell of the present invention under conditions conducive for productionof the polypeptide; and (b) recovering the polypeptide.

The host cells are cultivated in a nutrient medium suitable forproduction of the polypeptide using methods well known in the art. Forexample, the cell may be cultivated by shake flask cultivation, andsmall-scale or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors performed in a suitable medium and under conditions allowingthe polypeptide to be expressed and/or isolated. The cultivation takesplace in a suitable nutrient medium comprising carbon and nitrogensources and inorganic salts, using procedures known in the art. Suitablemedia are available from commercial suppliers or may be preparedaccording to published compositions (e.g., in catalogues of the AmericanType Culture Collection). If the polypeptide is secreted into thenutrient medium, the polypeptide can be recovered directly from themedium. If the polypeptide is not secreted, it can be recovered fromcell lysates.

The polypeptide may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate. For example, an enzyme assay may be used todetermine the activity of the polypeptide.

The polypeptide may be recovered using methods known in the art. Forexample, the polypeptide may be recovered from the nutrient medium byconventional procedures including, but not limited to, centrifugation,filtration, extraction, spray-drying, evaporation, or precipitation.

The polypeptide may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Jansonand Lars Ryden, editors, VCH Publishers, New York, 1989) to obtainsubstantially pure polypeptides.

In an alternative aspect, the polypeptide is not recovered, but rather ahost cell of the present invention expressing the polypeptide is used asa source of the polypeptide.

Plants

The present invention also relates to isolated plants, e.g., atransgenic plant, plant part, or plant cell, comprising an isolatedpolynucleotide of the present invention so as to express and produce thepolypeptide in recoverable quantities. The polypeptide may be recoveredfrom the plant or plant part. Alternatively, the plant or plant partcontaining the polypeptide may be used as such for improving the qualityof a food or feed, e.g., improving nutritional value, palatability, andrheological properties, or to destroy an antinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilization of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seeds coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing a polypeptide may beconstructed in accordance with methods known in the art. In short, theplant or plant cell is constructed by incorporating one or more(several) expression constructs encoding a polypeptide into the planthost genome or chloroplast genome and propagating the resulting modifiedplant or plant cell into a transgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct thatcomprises a polynucleotide encoding a polypeptide operably linked withappropriate regulatory sequences required for expression of thepolynucleotide in the plant or plant part of choice. Furthermore, theexpression construct may comprise a selectable marker useful foridentifying host cells into which the expression construct has beenintegrated and DNA sequences necessary for introduction of the constructinto the plant in question (the latter depends on the DNA introductionmethod to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences, is determined, forexample, on the basis of when, where, and how the polypeptide is desiredto be expressed. For instance, the expression of the gene encoding apolypeptide may be constitutive or inducible, or may be developmental,stage or tissue specific, and the gene product may be targeted to aspecific tissue or plant part such as seeds or leaves. Regulatorysequences are, for example, described by Tague et al., 1988, PlantPhysiology 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, andthe rice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294; Christensen et al., 1992, Plant Mol. Biol. 18: 675-689; Zhanget al., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be,for example, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant Cell Physiol. 39: 885-889), a Vicia faba promoter fromthe legumin B4 and the unknown seed protein gene from Vicia faba (Conradet al., 1998, J. Plant Physiol. 152: 708-711), a promoter from a seedoil body protein (Chen et al., 1998, Plant Cell Physiol. 39: 935-941),the storage protein napA promoter from Brassica napus, or any other seedspecific promoter known in the art, e.g., as described in WO 91/14772.Furthermore, the promoter may be a leaf specific promoter such as therbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiol.102: 991-1000), the chlorella virus adenine methyltransferase genepromoter (Mitra and Higgins, 1994, Plant Mol. Biol. 26: 85-93), the aldPgene promoter from rice (Kagaya et al., 1995, Mol. Gen. Genet. 248:668-674), or a wound inducible promoter such as the potato pin2 promoter(Xu et al., 1993, Plant Mol. Biol. 22: 573-588). Likewise, the promotermay inducible by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g., ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a polypeptide in the plant. For instance, the promoterenhancer element may be an intron that is placed between the promoterand the polynucleotide encoding a polypeptide. For instance, Xu et al.,1993, supra, disclose the use of the first intron of the rice actin 1gene to enhance expression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Agrobacterium tumefaciens-mediated gene transfer is a method forgenerating transgenic dicots (for a review, see Hooykas andSchilperoort, 1992, Plant Mol. Biol. 19: 15-38) and can also be used fortransforming monocots, although other transformation methods are oftenused for these plants. A method for generating transgenic monocots isparticle bombardment (microscopic gold or tungsten particles coated withthe transforming DNA) of embryonic calli or developing embryos(Christou, 1992, Plant J. 2: 275-281; Shimamoto, 1994, Curr. Opin.Biotechnol. 5: 158-162; Vasil et al., 1992, Bio/Technology 10: 667-674).An alternative method for transformation of monocots is based onprotoplast transformation as described by Omirulleh et al., 1993, PlantMol. Biol. 21: 415-428. Additional transformation methods for use inaccordance with the present disclosure include those described in U.S.Pat. Nos. 6,395,966 and 7,151,204 (both of which are herein incorporatedby reference in their entirety).

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using, forexample, co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

In addition to direct transformation of a particular plant genotype witha construct prepared according to the present invention, transgenicplants may be made by crossing a plant having the construct to a secondplant lacking the construct. For example, a construct encoding apolypeptide can be introduced into a particular plant variety bycrossing, without the need for ever directly transforming a plant ofthat given variety. Therefore, the present invention encompasses notonly a plant directly regenerated from cells which have been transformedin accordance with the present invention, but also the progeny of suchplants. As used herein, progeny may refer to the offspring of anygeneration of a parent plant prepared in accordance with the presentinvention. Such progeny may include a DNA construct prepared inaccordance with the present invention, or a portion of a DNA constructprepared in accordance with the present invention. Crossing results inthe introduction of a transgene into a plant line by cross pollinating astarting line with a donor plant line. Non-limiting examples of suchsteps are further articulated in U.S. Pat. No. 7,151,204.

Plants may be generated through a process of backcross conversion. Forexample, plants include plants referred to as a backcross convertedgenotype, line, inbred, or hybrid.

Genetic markers may be used to assist in the introgression of one ormore transgenes of the invention from one genetic background intoanother. Marker assisted selection offers advantages relative toconventional breeding in that it can be used to avoid errors caused byphenotypic variations. Further, genetic markers may provide dataregarding the relative degree of elite germplasm in the individualprogeny of a particular cross. For example, when a plant with a desiredtrait which otherwise has a non-agronomically desirable geneticbackground is crossed to an elite parent, genetic markers may be used toselect progeny which not only possess the trait of interest, but alsohave a relatively large proportion of the desired germplasm. In thisway, the number of generations required to introgress one or more traitsinto a particular genetic background is minimized.

The present invention also relates to methods of producing a polypeptideof the present invention comprising: (a) cultivating a transgenic plantor a plant cell comprising a polynucleotide encoding the polypeptideunder conditions conducive for production of the polypeptide; and (b)recovering the polypeptide.

Compositions

The present invention also relates to enzyme compositions comprising apolypeptide of the present invention. Preferably, the compositions areenriched in such a polypeptide. The term “enriched” indicates that thecellobiohydrolase activity of the composition has been increased, e.g.,with an enrichment factor of at least 1.1.

The composition may comprise a polypeptide of the present invention asthe major enzymatic component, e.g., a mono-component composition.Alternatively, the composition may comprise multiple enzymaticactivities, such as one or more (several) enzymes selected from thegroup consisting of a cellulase, a hemicellulase, an expansin, anesterase, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, aprotease, and a swollenin.

In a preferred embodiment the enzyme composition comprises at least thecellobiohydolase of the invention, at least one endoglucanase, at leastone beta-glucosidase and at least one GH61 polypeptide havingcellulolytic enhancing activity.

The polypeptide compositions may be prepared in accordance with methodsknown in the art and may be in the form of a liquid or a drycomposition. For instance, the polypeptide composition may be in theform of a granulate or a microgranulate. The polypeptide to be includedin the composition may be stabilized in accordance with methods known inthe art.

The enzyme compositions can comprise any protein that is useful indegrading or converting the cellulosic material.

In one aspect, the enzyme composition comprises or further comprises oneor more (several) proteins selected from the group consisting of acellulase, a GH61 polypeptide having cellulolytic enhancing activity, ahemicellulase, an expansin, an esterase, a laccase, a ligninolyticenzyme, a pectinase, a peroxidase, a protease, and a swollenin. Inanother aspect, the cellulase is preferably one or more (several)enzymes selected from the group consisting of an endoglucanase, anadditional cellobiohydrolase, and a beta-glucosidase. In another aspect,the hemicellulase is preferably one or more (several) enzymes selectedfrom the group consisting of an acetylmannan esterase, an acetyxylanesterase, an arabinanase, an arabinofuranosidase, a coumaric acidesterase, a feruloyl esterase, a galactosidase, a glucuronidase, aglucuronoyl esterase, a mannanase, a mannosidase, a xylanase, and axylosidase.

In another aspect, the enzyme composition comprises one or more(several) cellulolytic enzymes. In another aspect, the enzymecomposition comprises or further comprises one or more (several)hemicellulolytic enzymes. In another aspect, the enzyme compositioncomprises one or more (several) cellulolytic enzymes and one or more(several) hemicellulolytic enzymes. In another aspect, the enzymecomposition comprises one or more (several) enzymes selected from thegroup of cellulolytic enzymes and hemicellulolytic enzymes. In anotheraspect, the enzyme composition comprises an endoglucanase. In anotheraspect, the enzyme composition comprises a cellobiohydrolase. In anotheraspect, the enzyme composition comprises a beta-glucosidase. In anotheraspect, the enzyme composition comprises a GH61 polypeptide havingcellulolytic enhancing activity. In another aspect, the enzymecomposition comprises an endoglucanase and a cellobiohydrolase. Inanother aspect, the enzyme composition comprises an endoglucanase and abeta-glucosidase. In another aspect, the enzyme composition comprises acellobiohydrolase and a beta-glucosidase. In another aspect, the enzymecomposition comprises an endoglucanase, a cellobiohydrolase, and abeta-glucosidase. In another aspect, the enzyme composition comprises anendoglucanase, a cellobiohydrolase, a beta-glucosidase, and a GH61polypeptide having cellulolytic enhancing activity. In another aspect,the enzyme composition comprises an acetylmannan esterase. In anotheraspect, the enzyme composition comprises an acetyxylan esterase. Inanother aspect, the enzyme composition comprises an arabinanase (e.g.,alpha-L-arabinanase). In another aspect, the enzyme compositioncomprises an arabinofuranosidase (e.g., alpha-L-arabinofuranosidase). Inanother aspect, the enzyme composition comprises a coumaric acidesterase. In another aspect, the enzyme composition comprises a feruloylesterase. In another aspect, the enzyme composition comprises agalactosidase (e.g., alpha-galactosidase and/or beta-galactosidase). Inanother aspect, the enzyme composition comprises a glucuronidase (e.g.,alpha-D-glucuronidase). In another aspect, the enzyme compositioncomprises a glucuronoyl esterase. In another aspect, the enzymecomposition comprises a mannanase. In another aspect, the enzymecomposition comprises a mannosidase (e.g., beta-mannosidase). In anotheraspect, the enzyme composition comprises a xylanase. In a preferredaspect, the xylanase is a Family 10 xylanase. In another aspect, theenzyme composition comprises a xylosidase (e.g., beta-xylosidase). Inanother aspect, the enzyme composition comprises an expansin. In anotheraspect, the enzyme composition comprises an esterase. In another aspect,the enzyme composition comprises a laccase. In another aspect, theenzyme composition comprises a ligninolytic enzyme. In a preferredaspect, the ligninolytic enzyme is a manganese peroxidase. In anotherpreferred aspect, the ligninolytic enzyme is a lignin peroxidase. Inanother preferred aspect, the ligninolytic enzyme is a H₂O₂-producingenzyme. In another aspect, the enzyme composition comprises a pectinase.In another aspect, the enzyme composition comprises a peroxidase. Inanother aspect, the enzyme composition comprises a protease. In anotheraspect, the enzyme composition comprises a swollenin.

In the methods of the present invention, the enzyme(s) can be addedprior to or during fermentation, e.g., during saccharification or duringor after propagation of the fermenting microorganism(s).

One or more (several) components of the enzyme composition may bewild-type proteins, recombinant proteins, or a combination of wild-typeproteins and recombinant proteins. For example, one or more (several)components may be native proteins of a cell, which is used as a hostcell to express recombinantly one or more (several) other components ofthe enzyme composition. One or more (several) components of the enzymecomposition may be produced as monocomponents, which are then combinedto form the enzyme composition. The enzyme composition may be acombination of multicomponent and monocomponent protein preparations.

The enzymes used in the methods of the present invention may be in anyform suitable for use, such as, for example, a crude fermentation brothwith or without cells removed, a cell lysate with or without cellulardebris, a semi-purified or purified enzyme preparation, or a host cellas a source of the enzymes. The enzyme composition may be a dry powderor granulate, a non-dusting granulate, a liquid, a stabilized liquid, ora stabilized protected enzyme. Liquid enzyme preparations may, forinstance, be stabilized by adding stabilizers such as a sugar, a sugaralcohol or another polyol, and/or lactic acid or another organic acidaccording to established processes.

The optimum amounts of the enzymes and a polypeptide havingcellobiohydrolase activity depend on several factors including, but notlimited to, the mixture of component cellulolytic enzymes, thecellulosic material, the concentration of cellulosic material, thepretreatment(s) of the cellulosic material, temperature, time, pH, andinclusion of fermenting organism (e.g., yeast for SimultaneousSaccharification and Fermentation).

In a preferred aspect, an effective amount of cellulolytic enzyme to thecellulosic material is about 0.5 to about 50 mg, preferably about 0.5 toabout 40 mg, more preferably about 0.5 to about 25 mg, more preferablyabout 0.75 to about 20 mg, more preferably about 0.75 to about 15 mg,even more preferably about 0.5 to about 10 mg, and most preferably about2.5 to about 10 mg per g of the cellulosic material.

In another preferred aspect, an effective amount of a polypeptide havingcellobiohydrolase activity to the cellulosic material is about 0.01 toabout 50.0 mg, preferably about 0.01 to about 40 mg, more preferablyabout 0.01 to about 30 mg, more preferably about 0.01 to about 20 mg,more preferably about 0.01 to about 10 mg, more preferably about 0.01 toabout 5 mg, more preferably about 0.025 to about 1.5 mg, more preferablyabout 0.05 to about 1.25 mg, more preferably about 0.075 to about 1.25mg, more preferably about 0.1 to about 1.25 mg, even more preferablyabout 0.15 to about 1.25 mg, and most preferably about 0.25 to about 1.0mg per g of the cellulosic material.

In another preferred aspect, an effective amount of a polypeptide havingcellobiohydrolase activity to cellulolytic enzyme is about 0.005 toabout 1.0 g, preferably about 0.01 to about 1.0 g, more preferably about0.15 to about 0.75 g, more preferably about 0.15 to about 0.5 g, morepreferably about 0.1 to about 0.5 g, even more preferably about 0.1 toabout 0.25 g, and most preferably about 0.05 to about 0.2 g per g ofcellulolytic enzyme.

The polypeptides having cellulolytic enzyme activity or hemicellulolyticenzyme activity as well as other proteins/polypeptides useful in thedegradation of the cellulosic material, e.g., polypeptides havingcellulolytic enhancing activity (hereinafter “polypeptides having enzymeactivity”) can be derived or obtained from any suitable origin,including, bacterial, fungal, yeast, plant, or mammalian origin. Theterm “obtained” means herein that the enzyme may have been isolated froman organism that naturally produces the enzyme as a native enzyme. Theterm “obtained” also means herein that the enzyme may have been producedrecombinantly in a host organism employing methods described herein,wherein the recombinantly produced enzyme is either native or foreign tothe host organism or has a modified amino acid sequence, e.g., havingone or more (several) amino acids that are deleted, inserted and/orsubstituted, i.e., a recombinantly produced enzyme that is a mutantand/or a fragment of a native amino acid sequence or an enzyme producedby nucleic acid shuffling processes known in the art. Encompassed withinthe meaning of a native enzyme are natural variants and within themeaning of a foreign enzyme are variants obtained recombinantly, such asby site-directed mutagenesis or shuffling.

One or more (several) components of the enzyme composition may be arecombinant component, i.e., produced by cloning of a DNA sequenceencoding the single component and subsequent cell transformed with theDNA sequence and expressed in a host (see, for example, WO 91/17243 andWO 91/17244). The host is preferably a heterologous host (enzyme isforeign to host), but the host may under certain conditions also be ahomologous host (enzyme is native to host). Monocomponent cellulolyticproteins may also be prepared by purifying such a protein from afermentation broth.

In one aspect, the one or more (several) cellulolytic enzymes comprise acommercial cellulolytic enzyme preparation. Examples of commercialcellulolytic enzyme preparations suitable for use in the presentinvention include, for example, CELLIC™ CTec (Novozymes A/S), CELLIC™CTec2 (Novozymes A/S), CELLUCLAST™ (Novozymes A/S), NOVOZYM™ 188(Novozymes A/S), CELLUZYME™ (Novozymes A/S), CEREFLO™ (Novozymes A/S),and ULTRAFLO™ (Novozymes A/S), ACCELERASE™ (Genencor Int.), LAMINEX™(Genencor Int.), SPEZYME™ CP (Genencor Int.), ROHAMENT™ 7069 W (RöhmGmbH), FIBREZYME® LDI (Dyadic International, Inc.), FIBREZYME® LBR(Dyadic International, Inc.), or VISCOSTAR® 150 L (Dyadic International,Inc.). The cellulase enzymes are added in amounts effective from about0.001 to about 5.0 wt % of solids, more preferably from about 0.025 toabout 4.0 wt % of solids, and most preferably from about 0.005 to about2.0 wt % of solids.

Examples of bacterial endoglucanases that can be used in the methods ofthe present invention, include, but are not limited to, an Acidothermuscellulolyticus endoglucanase (WO 91/05039; WO 93/15186; U.S. Pat. No.5,275,944; WO 96/02551; U.S. Pat. No. 5,536,655, WO 00/70031, WO05/093050); Thermobifida fusca endoglucanase III (WO 05/093050); andThermobifida fusca endoglucanase V (WO 05/093050).

Examples of fungal endoglucanases that can be used in the presentinvention include, but are not limited to, a Trichoderma reeseiendoglucanase I (Penttila et al., 1986, Gene 45: 253-263; Trichodermareesei Cel7B endoglucanase I; GENBANK™ accession no. M15665);Trichoderma reesei endoglucanase II (Saloheimo, et al., 1988, Gene63:11-22; Trichoderma reesei Cel5A endoglucanase II; GENBANK™ accessionno. M19373); Trichoderma reesei endoglucanase III (Okada et al., 1988,Appl. Environ. Microbiol. 64: 555-563; GENBANK™ accession no. AB003694);Trichoderma reesei endoglucanase V (Saloheimo et al., 1994, MolecularMicrobiology 13: 219-228; GENBANK™ accession no. Z33381); Aspergillusaculeatus endoglucanase (Ooi et al., 1990, Nucleic Acids Research 18:5884); Aspergillus kawachii endoglucanase (Sakamoto et al., 1995,Current Genetics 27: 435-439); Erwinia carotovara endoglucanase(Saarilahti et al., 1990, Gene 90: 9-14); Fusarium oxysporumendoglucanase (GENBANK™ accession no. L29381); Humicola grisea var.thermoidea endoglucanase (GENBANK™ accession no. AB003107); Melanocarpusalbomyces endoglucanase (GENBANK™ accession no. MAL515703); Neurosporacrassa endoglucanase (GENBANK™ accession no. XM_324477); Humicolainsolens endoglucanase V; Myceliophthora thermophila CBS 117.65endoglucanase; basidiomycete CBS 495.95 endoglucanase; basidiomycete CBS494.95 endoglucanase; Thielavia terrestris NRRL 8126 CEL6Bendoglucanase; Thielavia terrestris NRRL 8126 CEL6C endoglucanase;Thielavia terrestris NRRL 8126 CEL7C endoglucanase; Thielavia terrestrisNRRL 8126 CEL7E endoglucanase; Thielavia terrestris NRRL 8126 CEL7Fendoglucanase; Cladorrhinum foecundissimum ATCC 62373 CEL7Aendoglucanase; and Trichoderma reesei strain No. VTT-D-80133endoglucanase (GENBANK™ accession no. M15665).

Examples of additional cellobiohydrolases useful in hydrolysis ofbiomass include, but are not limited to, Trichoderma reeseicellobiohydrolase I; Trichoderma reesei cellobiohydrolase II; Humicolainsolens cellobiohydrolase I; Myceliophthora thermophilacellobiohydrolase II; Thielavia terrestris cellobiohydrolase II (CEL6A);Chaetomium thermophilum cellobiohydrolase I; and Chaetomium thermophilumcellobiohydrolase II.

Examples of beta-glucosidases useful in the present invention include,but are not limited to, Aspergillus oryzae beta-glucosidase; Aspergillusfumigatus beta-glucosidase; Penicillium brasilianum IBT 20888beta-glucosidase; Aspergillus niger beta-glucosidase; and Aspergillusaculeatus beta-glucosidase.

The Aspergillus oryzae polypeptide having beta-glucosidase activity canbe obtained according to WO 2002/095014. The Aspergillus fumigatuspolypeptide having beta-glucosidase activity can be obtained accordingto WO 2005/047499. The Penicillium brasilianum polypeptide havingbeta-glucosidase activity can be obtained according to WO 2007/019442.The Aspergillus niger polypeptide having beta-glucosidase activity canbe obtained according to Dan et al., 2000, J. Biol. Chem. 275:4973-4980. The Aspergillus aculeatus polypeptide having beta-glucosidaseactivity can be obtained according to Kawaguchi et al., 1996, Gene 173:287-288.

The beta-glucosidase may be a fusion protein. In one aspect, thebeta-glucosidase is the Aspergillus oryzae beta-glucosidase variant BGfusion protein or the Aspergillus oryzae beta-glucosidase fusion proteinobtained according to WO 2008/057637.

Other useful endoglucanases, cellobiohydrolases, and beta-glucosidasesare disclosed in numerous Glycosyl Hydrolase families using theclassification according to Henrissat, 1991, A classification ofglycosyl hydrolases based on amino-acid sequence similarities, Biochem.J. 280: 309-316, and Henrissat and Bairoch, 1996, Updating thesequence-based classification of glycosyl hydrolases, Biochem. J. 316:695-696.

Other cellulolytic enzymes that may be used in the present invention aredescribed in EP 495,257, EP 531,315, EP 531,372, WO 89/09259, WO94/07998, WO 95/24471, WO 96/11262, WO 96/29397, WO 96/034108, WO97/14804, WO 98/08940, WO 98/012307, WO 98/13465, WO 98/015619, WO98/015633, WO 98/028411, WO 99/06574, WO 99/10481, WO 99/025846, WO99/025847, WO 99/031255, WO 2000/009707, WO 2002/050245, WO2002/0076792, WO 2002/101078, WO 2003/027306, WO 2003/052054, WO2003/052055, WO 2003/052056, WO 2003/052057, WO 2003/052118, WO2004/016760, WO 2004/043980, WO 2004/048592, WO 2005/001065, WO2005/028636, WO 2005/093050, WO 2005/093073, WO 2006/074005, WO2006/117432, WO 2007/071818, WO 2007/071820, WO 2008/008070, WO2008/008793, U.S. Pat. Nos. 4,435,307, 5,457,046, 5,648,263, 5,686,593,5,691,178, 5,763,254, and 5,776,757.

In the methods of the present invention, any GH61 polypeptide havingcellulolytic enhancing activity can be used.

Examples of polypeptides having cellulolytic enhancing activity usefulin the methods of the present invention include, but are not limited to,polypeptides having cellulolytic enhancing activity from Thielaviaterrestris (WO 2005/074647); polypeptides having cellulolytic enhancingactivity from Thermoascus aurantiacus (WO 2005/074656); polypeptideshaving cellulolytic enhancing activity from Trichoderma reesei (WO2007/089290); and polypeptides having cellulolytic enhancing activityfrom Myceliophthora thermophila (WO 2009/085935; WO 2009/085859; WO2009/085864; WO 2009/085868).

In one aspect, the one or more (several) hemicellulolytic enzymescomprise a commercial hemicellulolytic enzyme preparation. Examples ofcommercial hemicellulolytic enzyme preparations suitable for use in thepresent invention include, for example, SHEARZYME™ (Novozymes A/S),CELLIC™ HTec (Novozymes A/S), CELLIC™ HTec2 (Novozymes A/S), VISCOZYME®(Novozymes A/S), ULTRAFLO® (Novozymes A/S), PULPZYME® HC (NovozymesA/S), MULTIFECT® Xylanase (Genencor), ECOPULP® TX-200A (AB Enzymes), HSP6000 Xylanase (DSM), DEPOL™ 333P (Biocatalysts Limit, Wales, UK), DEPOL™740 L. (Biocatalysts Limit, Wales, UK), and DEPOL™ 762P (BiocatalystsLimit, Wales, UK).

Examples of xylanases useful in the methods of the present inventioninclude, but are not limited to, Aspergillus aculeatus xylanase(GeneSeqP:AAR63790; WO 94/21785), Aspergillus fumigatus xylanases (WO2006/078256), and Thielavia terrestris NRRL 8126 xylanases (WO2009/079210).

Examples of beta-xylosidases useful in the methods of the presentinvention include, but are not limited to, Trichoderma reeseibeta-xylosidase (UniProtKB/TrEMBL accession number Q92458), Talaromycesemersonii (SwissProt accession number Q8X212), and Neurospora crassa(SwissProt accession number Q7SOW4).

Examples of acetylxylan esterases useful in the methods of the presentinvention include, but are not limited to, Hypocrea jecorina acetylxylanesterase (WO 2005/001036), Neurospora crassa acetylxylan esterase(UniProt accession number q7s259), Thielavia terrestris NRRL 8126acetylxylan esterase (WO 2009/042846), Chaetomium globosum acetylxylanesterase (Uniprot accession number Q2GWX4), Chaetomium gracileacetylxylan esterase (GeneSeqP accession number AAB82124), Phaeosphaerianodorum acetylxylan esterase (Uniprot accession number QOUHJ1), andHumicola insolens DSM 1800 acetylxylan esterase (WO 2009/073709).

Examples of ferulic acid esterases useful in the methods of the presentinvention include, but are not limited to, Humicola insolens DSM 1800feruloyl esterase (WO 2009/076122), Neurospora crassa feruloyl esterase(UniProt accession number Q9HGR3), and Neosartorya fischeri feruloylesterase (UniProt Accession number A1D9T4).

Examples of arabinofuranosidases useful in the methods of the presentinvention include, but are not limited to, Humicola insolens DSM 1800arabinofuranosidase (WO 2009/073383) and Aspergillus nigerarabinofuranosidase (GeneSeqP accession number AAR94170).

Examples of alpha-glucuronidases useful in the methods of the presentinvention include, but are not limited to, Aspergillus clavatusalpha-glucuronidase (UniProt accession number alcc12), Trichodermareesei alpha-glucuronidase (Uniprot accession number Q99024),Talaromyces emersonii alpha-glucuronidase (UniProt accession numberQ8X211), Aspergillus niger alpha-glucuronidase (Uniprot accession numberQ96WX9), Aspergillus terreus alpha-glucuronidase (SwissProt accessionnumber QOCJP9), and Aspergillus fumigatus alpha-glucuronidase (SwissProtaccession number Q4WW45).

The enzymes and proteins used in the methods of the present inventionmay be produced by fermentation of the above-noted microbial strains ona nutrient medium containing suitable carbon and nitrogen sources andinorganic salts, using procedures known in the art (see, e.g., Bennett,J. W. and LaSure, L. (eds.), More Gene Manipulations in Fungi, AcademicPress, C A, 1991). Suitable media are available from commercialsuppliers or may be prepared according to published compositions (e.g.,in catalogues of the American Type Culture Collection). Temperatureranges and other conditions suitable for growth and enzyme productionare known in the art (see, e.g., Bailey, J. E., and Ollis, D. F.,Biochemical Engineering Fundamentals, McGraw-Hill Book Company, N Y,1986)

The fermentation can be any method of cultivation of a cell resulting inthe expression or isolation of an enzyme or protein. Fermentation may,therefore, be understood as comprising shake flask cultivation, orsmall- or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors performed in a suitable medium and under conditions allowingthe enzyme to be expressed or isolated. The resulting enzymes producedby the methods described above may be recovered from the fermentationmedium and purified by conventional procedures.

Examples are given below of preferred uses of the polypeptidecompositions of the invention. The dosage of the polypeptide compositionof the invention and other conditions under which the composition isused may be determined on the basis of methods known in the art.

Uses

The present invention is also directed to the following methods forusing the polypeptides having cellobiohydrolase activity, orcompositions thereof.

The present invention also relates to methods for degrading orconverting a cellulosic material, comprising: treating the cellulosicmaterial with an enzyme composition in the presence of a polypeptidehaving cellobiohydrolase activity of the present invention. In oneaspect the cellulosic material is pretreated. In another aspect, themethod further comprises recovering the degraded or converted cellulosicmaterial. Soluble products of degradation or conversion of thecellulosic material can be separated from the insoluble cellulosicmaterial using technology well known in the art such as, for example,centrifugation, filtration, and gravity settling.

The present invention also relates to methods of producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with an enzyme composition in the presence of a polypeptidehaving cellobiohydrolase activity of the present invention; (b)fermenting the saccharified cellulosic material with one or more(several) fermenting microorganisms to produce the fermentation product;and (c) recovering the fermentation product from the fermentation.

The present invention also relates to methods of fermenting a cellulosicmaterial, comprising: fermenting the cellulosic material with one ormore (several) fermenting microorganisms, wherein the cellulosicmaterial is saccharified with an enzyme composition in the presence of apolypeptide having cellobiohydrolase activity of the present invention.In one aspect, the fermenting of the cellulosic material produces afermentation product. In another aspect, the method further comprisesrecovering the fermentation product from the fermentation.

The methods of the present invention can be used to saccharify thecellulosic material to fermentable sugars and convert the fermentablesugars to many useful substances, e.g., fuel, potable ethanol, and/orfermentation products (e.g., acids, alcohols, ketones, gases, and thelike). The production of a desired fermentation product from thecellulosic material typically involves pretreatment, enzymatichydrolysis (saccharification), and fermentation.

The processing of the cellulosic material according to the presentinvention can be accomplished using processes conventional in the art.Moreover, the methods of the present invention can be implemented usingany conventional biomass processing apparatus configured to operate inaccordance with the invention.

Hydrolysis (saccharification) and fermentation, separate orsimultaneous, include, but are not limited to, separate hydrolysis andfermentation (SHF); simultaneous saccharification and fermentation(SSF); simultaneous saccharification and cofermentation (SSCF); hybridhydrolysis and fermentation (HHF); separate hydrolysis andco-fermentation (SHCF); hybrid hydrolysis and co-fermentation (HHCF);and direct microbial conversion (DMC). SHF uses separate process stepsto first enzymatically hydrolyze the cellulosic material to fermentablesugars, e.g., glucose, cellobiose, cellotriose, and pentose sugars, andthen ferment the fermentable sugars to ethanol. In SSF, the enzymatichydrolysis of the cellulosic material and the fermentation of sugars toethanol are combined in one step (Philippidis, G. P., 1996, Cellulosebioconversion technology, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212). SSCF involves the cofermentation of multiple sugars (Sheehan,J., and Himmel, M., 1999, Enzymes, energy and the environment: Astrategic perspective on the U.S. Department of Energy's research anddevelopment activities for bioethanol, Biotechnol. Prog. 15: 817-827).HHF involves a separate hydrolysis step, and in addition a simultaneoussaccharification and hydrolysis step, which can be carried out in thesame reactor. The steps in an HHF process can be carried out atdifferent temperatures, i.e., high temperature enzymaticsaccharification followed by SSF at a lower temperature that thefermentation strain can tolerate. DMC combines all three processes(enzyme production, hydrolysis, and fermentation) in one or more(several) steps where the same organism is used to produce the enzymesfor conversion of the cellulosic material to fermentable sugars and toconvert the fermentable sugars into a final product (Lynd et al., 2002,Microbial cellulose utilization: Fundamentals and biotechnology,Microbiol. Mol. Biol. Reviews 66: 506-577). It is understood herein thatany method known in the art comprising pretreatment, enzymatichydrolysis (saccharification), fermentation, or a combination thereof,can be used in the practicing the methods of the present invention.

A conventional apparatus can include a fed-batch stirred reactor, abatch stirred reactor, a continuous flow stirred reactor withultrafiltration, and/or a continuous plug-flow column reactor (Corazzaet al., 2003, Optimal control in fed-batch reactor for the cellobiosehydrolysis, Acta Scientiarum. Technology 25: 33-38; Gusakov andSinitsyn, 1985, Kinetics of the enzymatic hydrolysis of cellulose: 1. Amathematical model for a batch reactor process, Enz. Microb. Technol. 7:346-352), an attrition reactor (Ryu and Lee, 1983, Bioconversion ofwaste cellulose by using an attrition bioreactor, Biotechnol. Bioeng.25: 53-65), or a reactor with intensive stirring induced by anelectromagnetic field (Gusakov et al., 1996, Enhancement of enzymaticcellulose hydrolysis using a novel type of bioreactor with intensivestirring induced by electromagnetic field, Appl. Biochem. Biotechnol.56: 141-153). Additional reactor types include: fluidized bed, upflowblanket, immobilized, and extruder type reactors for hydrolysis and/orfermentation.

Pretreatment. In practicing the methods of the present invention, anypretreatment process known in the art can be used to disrupt plant cellwall components of the cellulosic material (Chandra et al., 2007,Substrate pretreatment: The key to effective enzymatic hydrolysis oflignocellulosics? Adv. Biochem. Engin./Biotechnol. 108: 67-93; Galbe andZacchi, 2007, Pretreatment of lignocellulosic materials for efficientbioethanol production, Adv. Biochem. Engin./Biotechnol. 108: 41-65;Hendriks and Zeeman, 2009, Pretreatments to enhance the digestibility oflignocellulosic biomass, Bioresource Technol. 100: 10-18; Mosier et al.,2005, Features of promising technologies for pretreatment oflignocellulosic biomass, Bioresource Technol. 96: 673-686; Taherzadehand Karimi, 2008, Pretreatment of lignocellulosic wastes to improveethanol and biogas production: A review, Int. J. of Mol. Sci. 9:1621-1651; Yang and Wyman, 2008, Pretreatment: the key to unlockinglow-cost cellulosic ethanol, Biofuels Bioproducts andBiorefining-Biofpr. 2: 26-40).

The cellulosic material can also be subjected to particle sizereduction, pre-soaking, wetting, washing, and/or conditioning prior topretreatment using methods known in the art.

Conventional pretreatments include, but are not limited to, steampretreatment (with or without explosion), dilute acid pretreatment, hotwater pretreatment, alkaline pretreatment, lime pretreatment, wetoxidation, wet explosion, ammonia fiber explosion, organosolvpretreatment, and biological pretreatment. Additional pretreatmentsinclude ammonia percolation, ultrasound, electroporation, microwave,supercritical CO₂, supercritical H₂O, ozone, and gamma irradiationpretreatments.

The cellulosic material can be pretreated before hydrolysis and/orfermentation. Pretreatment is preferably performed prior to thehydrolysis. Alternatively, the pretreatment can be carried outsimultaneously with enzyme hydrolysis to release fermentable sugars,such as glucose, xylose, and/or cellobiose. In most cases thepretreatment step itself results in some conversion of biomass tofermentable sugars (even in absence of enzymes).

Steam Pretreatment: In steam pretreatment, the cellulosic material isheated to disrupt the plant cell wall components, including lignin,hemicellulose, and cellulose to make the cellulose and other fractions,e.g., hemicellulose, accessible to enzymes. The cellulosic material ispassed to or through a reaction vessel where steam is injected toincrease the temperature to the required temperature and pressure and isretained therein for the desired reaction time. Steam pretreatment ispreferably done at 140-230° C., more preferably 160-200° C., and mostpreferably 170-190° C., where the optimal temperature range depends onany addition of a chemical catalyst. Residence time for the steampretreatment is preferably 1-15 minutes, more preferably 3-12 minutes,and most preferably 4-10 minutes, where the optimal residence timedepends on temperature range and any addition of a chemical catalyst.Steam pretreatment allows for relatively high solids loadings, so thatthe cellulosic material is generally only moist during the pretreatment.The steam pretreatment is often combined with an explosive discharge ofthe material after the pretreatment, which is known as steam explosion,that is, rapid flashing to atmospheric pressure and turbulent flow ofthe material to increase the accessible surface area by fragmentation(Duff and Murray, 1996, Bioresource Technology 855: 1-33; Galbe andZacchi, 2002, Appl. Microbiol. Biotechnol. 59: 618-628; U.S. PatentApplication No. 2002/0164730). During steam pretreatment, hemicelluloseacetyl groups are cleaved and the resulting acid autocatalyzes partialhydrolysis of the hemicellulose to monosaccharides and oligosaccharides.Lignin is removed to only a limited extent.

A catalyst such as H₂SO₄ or SO₂ (typically 0.3 to 3% w/w) is often addedprior to steam pretreatment, which decreases the time and temperature,increases the recovery, and improves enzymatic hydrolysis (Ballesteroset al., 2006, Appl. Biochem. Biotechnol. 129-132: 496-508; Varga et al.,2004, Appl. Biochem. Biotechnol. 113-116: 509-523; Sassner et al., 2006,Enzyme Microb. Technol. 39: 756-762).

Chemical Pretreatment: The term “chemical treatment” refers to anychemical pretreatment that promotes the separation and/or release ofcellulose, hemicellulose, and/or lignin. Examples of suitable chemicalpretreatment processes include, for example, dilute acid pretreatment,lime pretreatment, wet oxidation, ammonia fiber/freeze explosion (AFEX),ammonia percolation (APR), and organosolv pretreatments.

In dilute acid pretreatment, the cellulosic material is mixed withdilute acid, typically H₂SO₄, and water to form a slurry, heated bysteam to the desired temperature, and after a residence time flashed toatmospheric pressure. The dilute acid pretreatment can be performed witha number of reactor designs, e.g., plug-flow reactors, counter-currentreactors, or continuous counter-current shrinking bed reactors (Duff andMurray, 1996, supra; Schell et al., 2004, Bioresource Technol. 91:179-188; Lee et al., 1999, Adv. Biochem. Eng. Biotechnol. 65: 93-115).

Several methods of pretreatment under alkaline conditions can also beused. These alkaline pretreatments include, but are not limited to, limepretreatment, wet oxidation, ammonia percolation (APR), and ammoniafiber/freeze explosion (AFEX).

Lime pretreatment is performed with calcium carbonate, sodium hydroxide,or ammonia at low temperatures of 85-150° C. and residence times from 1hour to several days (Wyman et al., 2005, Bioresource Technol. 96:1959-1966; Mosier et al., 2005, Bioresource Technol. 96: 673-686). WO2006/110891, WO 2006/110899, WO 2006/110900, and WO 2006/110901 disclosepretreatment methods using ammonia.

Wet oxidation is a thermal pretreatment performed typically at 180-200°C. for 5-15 minutes with addition of an oxidative agent such as hydrogenperoxide or over-pressure of oxygen (Schmidt and Thomsen, 1998,Bioresource Technol. 64: 139-151; Palonen et al., 2004, Appl. Biochem.Biotechnol. 117: 1-17; Varga et al., 2004, Biotechnol. Bioeng. 88:567-574; Martin et al., 2006, J. Chem. Technol. Biotechnol. 81:1669-1677). The pretreatment is performed at preferably 1-40% drymatter, more preferably 2-30% dry matter, and most preferably 5-20% drymatter, and often the initial pH is increased by the addition of alkalisuch as sodium carbonate.

A modification of the wet oxidation pretreatment method, known as wetexplosion (combination of wet oxidation and steam explosion), can handledry matter up to 30%. In wet explosion, the oxidizing agent isintroduced during pretreatment after a certain residence time. Thepretreatment is then ended by flashing to atmospheric pressure (WO2006/032282).

Ammonia fiber explosion (AFEX) involves treating the cellulosic materialwith liquid or gaseous ammonia at moderate temperatures such as 90-100°C. and high pressure such as 17-20 bar for 5-10 minutes, where the drymatter content can be as high as 60% (Gollapalli et al., 2002, Appl.Biochem. Biotechnol. 98: 23-35; Chundawat et al., 2007, Biotechnol.Bioeng. 96: 219-231; Alizadeh et al., 2005, Appl. Biochem. Biotechnol.121: 1133-1141; Teymouri et al., 2005, Bioresource Technol. 96:2014-2018). AFEX pretreatment results in the depolymerization ofcellulose and partial hydrolysis of hemicellulose. Lignin-carbohydratecomplexes are cleaved.

Organosolv pretreatment delignifies the cellulosic material byextraction using aqueous ethanol (40-60% ethanol) at 160-200° C. for30-60 minutes (Pan et al., 2005, Biotechnol. Bioeng. 90: 473-481; Pan etal., 2006, Biotechnol. Bioeng. 94: 851-861; Kurabi et al., 2005, Appl.Biochem. Biotechnol. 121: 219-230). Sulphuric acid is usually added as acatalyst. In organosolv pretreatment, the majority of hemicellulose isremoved.

Other examples of suitable pretreatment methods are described by Schellet al., 2003, Appl. Biochem. and Biotechnol. Vol. 105-108, p. 69-85, andMosier et al., 2005, Bioresource Technology 96: 673-686, and U.S.Published Application 2002/0164730.

In one aspect, the chemical pretreatment is preferably carried out as anacid treatment, and more preferably as a continuous dilute and/or mildacid treatment. The acid is typically sulfuric acid, but other acids canalso be used, such as acetic acid, citric acid, nitric acid, phosphoricacid, tartaric acid, succinic acid, hydrogen chloride, or mixturesthereof. Mild acid treatment is conducted in the pH range of preferably1-5, more preferably 1-4, and most preferably 1-3. In one aspect, theacid concentration is in the range from preferably 0.01 to 20 wt % acid,more preferably 0.05 to 10 wt % acid, even more preferably 0.1 to 5 wt %acid, and most preferably 0.2 to 2.0 wt % acid. The acid is contactedwith the cellulosic material and held at a temperature in the range ofpreferably 160-220° C., and more preferably 165-195° C., for periodsranging from seconds to minutes to, e.g., 1 second to 60 minutes.

In another aspect, pretreatment is carried out as an ammonia fiberexplosion step (AFEX pretreatment step).

In another aspect, pretreatment takes place in an aqueous slurry. Inpreferred aspects, the cellulosic material is present duringpretreatment in amounts preferably between 10-80 wt %, more preferablybetween 20-70 wt %, and most preferably between 30-60 wt %, such asaround 50 wt %. The pretreated cellulosic material can be unwashed orwashed using any method known in the art, e.g., washed with water.

Mechanical Pretreatment: The term “mechanical pretreatment” refers tovarious types of grinding or milling (e.g., dry milling, wet milling, orvibratory ball milling).

Physical Pretreatment: The term “physical pretreatment” refers to anypretreatment that promotes the separation and/or release of cellulose,hemicellulose, and/or lignin from the cellulosic material. For example,physical pretreatment can involve irradiation (e.g., microwaveirradiation), steaming/steam explosion, hydrothermolysis, andcombinations thereof.

Physical pretreatment can involve high pressure and/or high temperature(steam explosion). In one aspect, high pressure means pressure in therange of preferably about 300 to about 600 psi, more preferably about350 to about 550 psi, and most preferably about 400 to about 500 psi,such as around 450 psi. In another aspect, high temperature meanstemperatures in the range of about 100 to about 300° C., preferablyabout 140 to about 235° C. In a preferred aspect, mechanicalpretreatment is performed in a batch-process, steam gun hydrolyzersystem that uses high pressure and high temperature as defined above,e.g., a Sunds Hydrolyzer available from Sunds Defibrator AB, Sweden.

Combined Physical and Chemical Pretreatment: The cellulosic material canbe pretreated both physically and chemically. For instance, thepretreatment step can involve dilute or mild acid treatment and hightemperature and/or pressure treatment. The physical and chemicalpretreatments can be carried out sequentially or simultaneously, asdesired. A mechanical pretreatment can also be included.

Accordingly, in a preferred aspect, the cellulosic material is subjectedto mechanical, chemical, or physical pretreatment, or any combinationthereof, to promote the separation and/or release of cellulose,hemicellulose, and/or lignin.

Biological Pretreatment: The term “biological pretreatment” refers toany biological pretreatment that promotes the separation and/or releaseof cellulose, hemicellulose, and/or lignin from the cellulosic material.Biological pretreatment techniques can involve applyinglignin-solubilizing microorganisms (see, for example, Hsu, T.-A., 1996,Pretreatment of biomass, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212; Ghosh and Singh, 1993, Physicochemical and biologicaltreatments for enzymatic/microbial conversion of cellulosic biomass,Adv. Appl. Microbiol. 39: 295-333; McMillan, J. D., 1994, Pretreatinglignocellulosic biomass: a review, in Enzymatic Conversion of Biomassfor Fuels Production, Himmel, M. E., Baker, J. O., and Overend, R. P.,eds., ACS Symposium Series 566, American Chemical Society, Washington,D.C., chapter 15; Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T.,1999, Ethanol production from renewable resources, in Advances inBiochemical Engineering/Biotechnology, Scheper, T., ed., Springer-VerlagBerlin Heidelberg, Germany, 65: 207-241; Olsson and Hahn-Hagerdal, 1996,Fermentation of lignocellulosic hydrolysates for ethanol production,Enz. Microb. Tech. 18: 312-331; and Vallander and Eriksson, 1990,Production of ethanol from lignocellulosic materials: State of the art,Adv. Biochem. Eng./Biotechnol. 42: 63-95).

Saccharification. In the hydrolysis step, also known assaccharification, the cellulosic material, e.g., pretreated, ishydrolyzed to break down cellulose and alternatively also hemicelluloseto fermentable sugars, such as glucose, cellobiose, xylose, xylulose,arabinose, mannose, galactose, and/or soluble oligosaccharides. Thehydrolysis is performed enzymatically by an enzyme composition in thepresence of a polypeptide having cellobiohydrolase activity of thepresent invention. The enzymes of the compositions can be addedsequentially.

Enzymatic hydrolysis is preferably carried out in a suitable aqueousenvironment under conditions that can be readily determined by oneskilled in the art. In one aspect, hydrolysis is performed underconditions suitable for the activity of the enzyme(s), i.e., optimal forthe enzyme(s). The hydrolysis can be carried out as a fed batch orcontinuous process where the cellulosic material is fed gradually to,for example, an enzyme containing hydrolysis solution.

The saccharification is generally performed in stirred-tank reactors orfermentors under controlled pH, temperature, and mixing conditions.Suitable process time, temperature and pH conditions can readily bedetermined by one skilled in the art. For example, the saccharificationcan last up to 200 hours, but is typically performed for preferablyabout 12 to about 96 hours, more preferably about 16 to about 72 hours,and most preferably about 24 to about 48 hours. The temperature is inthe range of preferably about 25° C. to about 70° C., more preferablyabout 30° C. to about 65° C., and more preferably about 40° C. to 60°C., in particular about 50° C. The pH is in the range of preferablyabout 3 to about 8, more preferably about 3.5 to about 7, and mostpreferably about 4 to about 6, in particular about pH 5. The dry solidscontent is in the range of preferably about 5 to about 50 wt %, morepreferably about 10 to about 40 wt %, and most preferably about 20 toabout 30 wt %.

Fermentation. The fermentable sugars obtained from the hydrolyzedcellulosic material can be fermented by one or more (several) fermentingmicroorganisms capable of fermenting the sugars directly or indirectlyinto a desired fermentation product. “Fermentation” or “fermentationprocess” refers to any fermentation process or any process comprising afermentation step. Fermentation processes also include fermentationprocesses used in the consumable alcohol industry (e.g., beer and wine),dairy industry (e.g., fermented dairy products), leather industry, andtobacco industry. The fermentation conditions depend on the desiredfermentation product and fermenting organism and can easily bedetermined by one skilled in the art.

In the fermentation step, sugars, released from the cellulosic materialas a result of the pretreatment and enzymatic hydrolysis steps, arefermented to a product, e.g., ethanol, by a fermenting organism, such asyeast. Hydrolysis (saccharification) and fermentation can be separate orsimultaneous, as described herein.

Any suitable hydrolyzed cellulosic material can be used in thefermentation step in practicing the present invention. The material isgenerally selected based on the desired fermentation product, i.e., thesubstance to be obtained from the fermentation, and the processemployed, as is well known in the art.

The term “fermentation medium” is understood herein to refer to a mediumbefore the fermenting microorganism(s) is(are) added, such as, a mediumresulting from a saccharification process, as well as a medium used in asimultaneous saccharification and fermentation process (SSF).

“Fermenting microorganism” refers to any microorganism, includingbacterial and fungal organisms, suitable for use in a desiredfermentation process to produce a fermentation product. The fermentingorganism can be C₆ and/or C₅ fermenting organisms, or a combinationthereof. Both C₆ and C₅ fermenting organisms are well known in the art.Suitable fermenting microorganisms are able to ferment, i.e., convert,sugars, such as glucose, xylose, xylulose, arabinose, maltose, mannose,galactose, or oligosaccharides, directly or indirectly into the desiredfermentation product.

Examples of bacterial and fungal fermenting organisms producing ethanolare described by Lin et al., 2006, Appl. Microbiol. Biotechnol. 69:627-642.

Examples of fermenting microorganisms that can ferment C₆ sugars includebacterial and fungal organisms, such as yeast. Preferred yeast includesstrains of the Saccharomyces spp., preferably Saccharomyces cerevisiae.

Examples of fermenting organisms that can ferment C₅ sugars includebacterial and fungal organisms, such as some yeast. Preferred C₅fermenting yeast include strains of Pichia, preferably Pichia stipitis,such as Pichia stipitis CBS 5773; strains of Candida, preferably Candidaboidinii, Candida brassicae, Candida sheatae, Candida diddensii, Candidapseudotropicalis, or Candida utilis.

Other fermenting organisms include strains of Zymomonas, such asZymomonas mobilis; Hansenula, such as Hansenula anomala; Kluyveromyces,such as K. fragilis; Schizosaccharomyces, such as S. pombe; E. coli,especially E. coli strains that have been genetically modified toimprove the yield of ethanol; Clostridium, such as Clostridiumacetobutylicum, Chlostridium thermocellum, and Chlostridiumphytofermentans; Geobacillus sp.; Thermoanaerobacter, such asThermoanaerobacter saccharolyticum; and Bacillus, such as Bacilluscoagulans.

In a preferred aspect, the yeast is a Saccharomyces spp. In a morepreferred aspect, the yeast is Saccharomyces cerevisiae. In another morepreferred aspect, the yeast is Saccharomyces distaticus. In another morepreferred aspect, the yeast is Saccharomyces uvarum. In anotherpreferred aspect, the yeast is a Kluyveromyces. In another morepreferred aspect, the yeast is Kluyveromyces marxianus. In another morepreferred aspect, the yeast is Kluyveromyces fragilis. In anotherpreferred aspect, the yeast is a Candida. In another more preferredaspect, the yeast is Candida boidinii. In another more preferred aspect,the yeast is Candida brassicae. In another more preferred aspect, theyeast is Candida diddensii. In another more preferred aspect, the yeastis Candida pseudotropicalis. In another more preferred aspect, the yeastis Candida utilis. In another preferred aspect, the yeast is aClavispora. In another more preferred aspect, the yeast is Clavisporalusitaniae. In another more preferred aspect, the yeast is Clavisporaopuntiae. In another preferred aspect, the yeast is a Pachysolen. Inanother more preferred aspect, the yeast is Pachysolen tannophilus. Inanother preferred aspect, the yeast is a Pichia. In another morepreferred aspect, the yeast is a Pichia stipitis. In another preferredaspect, the yeast is a Bretannomyces. In another more preferred aspect,the yeast is Bretannomyces clausenii (Philippidis, G. P., 1996,Cellulose bioconversion technology, in Handbook on Bioethanol:Production and Utilization, Wyman, C. E., ed., Taylor & Francis,Washington, D.C., 179-212).

Bacteria that can efficiently ferment hexose and pentose to ethanolinclude, for example, Zymomonas mobilis, Clostridium acetobutylicum,Clostridium thermocellum, Chlostridium phytofermentans, Geobacillus sp.,Thermoanaerobacter saccharolyticum, and Bacillus coagulans (Philippidis,1996, supra).

In a preferred aspect, the bacterium is a Zymomonas. In a more preferredaspect, the bacterium is Zymomonas mobilis. In another preferred aspect,the bacterium is a Clostridium. In another more preferred aspect, thebacterium is Clostridium thermocellum.

Commercially available yeast suitable for ethanol production includes,e.g., ETHANOL RED™ yeast (Fermentis/Lesaffre, USA), FALI™ (Fleischmann'sYeast, USA), SUPERSTART™ and THERMOSACC™ fresh yeast (EthanolTechnology, WI, USA), BIOFERM™ AFT and XR (NABC—North AmericanBioproducts Corporation, GA, USA), GERT STRAND™ (Gert Strand AB,Sweden), and FERMIOL™ (DSM Specialties).

In a preferred aspect, the fermenting microorganism has been geneticallymodified to provide the ability to ferment pentose sugars, such asxylose utilizing, arabinose utilizing, and xylose and arabinoseco-utilizing microorganisms.

The cloning of heterologous genes into various fermenting microorganismshas led to the construction of organisms capable of converting hexosesand pentoses to ethanol (cofermentation) (Chen and Ho, 1993, Cloning andimproving the expression of Pichia stipitis xylose reductase gene inSaccharomyces cerevisiae, Appl. Biochem. Biotechnol. 39-40: 135-147; Hoet al., 1998, Genetically engineered Saccharomyces yeast capable ofeffectively cofermenting glucose and xylose, Appl. Environ. Microbiol.64: 1852-1859; Kotter and Ciriacy, 1993, Xylose fermentation bySaccharomyces cerevisiae, Appl. Microbiol. Biotechnol. 38: 776-783;Walfridsson et al., 1995, Xylose-metabolizing Saccharomyces cerevisiaestrains overexpressing the TKL1 and TALI genes encoding the pentosephosphate pathway enzymes transketolase and transaldolase, Appl.Environ. Microbiol. 61: 4184-4190; Kuyper et al., 2004, Minimalmetabolic engineering of Saccharomyces cerevisiae for efficientanaerobic xylose fermentation: a proof of principle, FEMS Yeast Research4: 655-664; Beall et al., 1991, Parametric studies of ethanol productionfrom xylose and other sugars by recombinant Escherichia coli, Biotech.Bioeng. 38: 296-303; Ingram et al., 1998, Metabolic engineering ofbacteria for ethanol production, Biotechnol. Bioeng. 58: 204-214; Zhanget al., 1995, Metabolic engineering of a pentose metabolism pathway inethanologenic Zymomonas mobilis, Science 267: 240-243; Deanda et al.,1996, Development of an arabinose-fermenting Zymomonas mobilis strain bymetabolic pathway engineering, Appl. Environ. Microbiol. 62: 4465-4470;WO 2003/062430, xylose isomerase).

In a preferred aspect, the genetically modified fermenting microorganismis Saccharomyces cerevisiae. In another preferred aspect, thegenetically modified fermenting microorganism is Zymomonas mobilis. Inanother preferred aspect, the genetically modified fermentingmicroorganism is Escherichia coli. In another preferred aspect, thegenetically modified fermenting microorganism is Klebsiella oxytoca. Inanother preferred aspect, the genetically modified fermentingmicroorganism is Kluyveromyces sp.

It is well known in the art that the organisms described above can alsobe used to produce other substances, as described herein.

The fermenting microorganism is typically added to the degradedlignocellulose or hydrolysate and the fermentation is performed forabout 8 to about 96 hours, such as about 24 to about 60 hours. Thetemperature is typically between about 26° C. to about 60° C., inparticular about 32° C. or 50° C., and at about pH 3 to about pH 8, suchas around pH 4-5, 6, or 7.

In a preferred aspect, the yeast and/or another microorganism is appliedto the degraded cellulosic material and the fermentation is performedfor about 12 to about 96 hours, such as typically 24-60 hours. In apreferred aspect, the temperature is preferably between about 20° C. toabout 60° C., more preferably about 25° C. to about 50° C., and mostpreferably about 32° C. to about 50° C., in particular about 32° C. or50° C., and the pH is generally from about pH 3 to about pH 7,preferably around pH 4-7. However, some fermenting organisms, e.g.,bacteria, have higher fermentation temperature optima. Yeast or anothermicroorganism is preferably applied in amounts of approximately 10⁵ to10¹², preferably from approximately 10⁷ to 10¹⁰, especiallyapproximately 2×10⁸ viable cell count per ml of fermentation broth.Further guidance in respect of using yeast for fermentation can be foundin, e.g., “The Alcohol Textbook” (Editors K. Jacques, T. P. Lyons and D.R. Kelsall, Nottingham University Press, United Kingdom 1999), which ishereby incorporated by reference.

For ethanol production, following the fermentation the fermented slurryis distilled to extract the ethanol. The ethanol obtained according tothe methods of the invention can be used as, e.g., fuel ethanol,drinking ethanol, i.e., potable neutral spirits, or industrial ethanol.

A fermentation stimulator can be used in combination with any of theprocesses described herein to further improve the fermentation process,and in particular, the performance of the fermenting microorganism, suchas, rate enhancement and ethanol yield. A “fermentation stimulator”refers to stimulators for growth of the fermenting microorganisms, inparticular, yeast. Preferred fermentation stimulators for growth includevitamins and minerals. Examples of vitamins include multivitamins,biotin, pantothenate, nicotinic acid, meso-inositol, thiamine,pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and VitaminsA, B, C, D, and E. See, for example, Alfenore et al., Improving ethanolproduction and viability of Saccharomyces cerevisiae by a vitaminfeeding strategy during fed-batch process, Springer-Verlag (2002), whichis hereby incorporated by reference. Examples of minerals includeminerals and mineral salts that can supply nutrients comprising P, K,Mg, S, Ca, Fe, Zn, Mn, and Cu.

Fermentation Products: A fermentation product can be any substancederived from the fermentation. The fermentation product can be, withoutlimitation, an alcohol (e.g., arabinitol, butanol, ethanol, glycerol,methanol, 1,3-propanediol, sorbitol, and xylitol); an organic acid(e.g., acetic acid, acetonic acid, adipic acid, ascorbic acid, citricacid, 2,5-diketo-D-gluconic acid, formic acid, fumaric acid, glucaricacid, gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionicacid, itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid,oxaloacetic acid, propionic acid, succinic acid, and xylonic acid); aketone (e.g., acetone); an amino acid (e.g., aspartic acid, glutamicacid, glycine, lysine, serine, and threonine); and a gas (e.g., methane,hydrogen (H₂), carbon dioxide (CO₂), and carbon monoxide (CO)). Thefermentation product can also be protein as a high value product.

In a preferred aspect, the fermentation product is an alcohol. It willbe understood that the term “alcohol” encompasses a substance thatcontains one or more hydroxyl moieties. In a more preferred aspect, thealcohol is arabinitol. In another more preferred aspect, the alcohol isbutanol. In another more preferred aspect, the alcohol is ethanol. Inanother more preferred aspect, the alcohol is glycerol. In another morepreferred aspect, the alcohol is methanol. In another more preferredaspect, the alcohol is 1,3-propanediol. In another more preferredaspect, the alcohol is sorbitol. In another more preferred aspect, thealcohol is xylitol. See, for example, Gong, C. S., Cao, N. J., Du, J.,and Tsao, G. T., 1999, Ethanol production from renewable resources, inAdvances in Biochemical Engineering/Biotechnology, Scheper, T., ed.,Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Silveira, M.M., and Jonas, R., 2002, The biotechnological production of sorbitol,Appl. Microbiol. Biotechnol. 59: 400-408; Nigam and Singh, 1995,Processes for fermentative production of xylitol—a sugar substitute,Process Biochemistry 30(2): 117-124; Ezeji et al., 2003, Production ofacetone, butanol and ethanol by Clostridium beijerinckii BA101 and insitu recovery by gas stripping, World Journal of Microbiology andBiotechnology 19 (6): 595-603.

In another preferred aspect, the fermentation product is an organicacid. In another more preferred aspect, the organic acid is acetic acid.In another more preferred aspect, the organic acid is acetonic acid. Inanother more preferred aspect, the organic acid is adipic acid. Inanother more preferred aspect, the organic acid is ascorbic acid. Inanother more preferred aspect, the organic acid is citric acid. Inanother more preferred aspect, the organic acid is 2,5-diketo-D-gluconicacid. In another more preferred aspect, the organic acid is formic acid.In another more preferred aspect, the organic acid is fumaric acid. Inanother more preferred aspect, the organic acid is glucaric acid. Inanother more preferred aspect, the organic acid is gluconic acid. Inanother more preferred aspect, the organic acid is glucuronic acid. Inanother more preferred aspect, the organic acid is glutaric acid. Inanother preferred aspect, the organic acid is 3-hydroxypropionic acid.In another more preferred aspect, the organic acid is itaconic acid. Inanother more preferred aspect, the organic acid is lactic acid. Inanother more preferred aspect, the organic acid is malic acid. Inanother more preferred aspect, the organic acid is malonic acid. Inanother more preferred aspect, the organic acid is oxalic acid. Inanother more preferred aspect, the organic acid is propionic acid. Inanother more preferred aspect, the organic acid is succinic acid. Inanother more preferred aspect, the organic acid is xylonic acid. See,for example, Chen and Lee, 1997, Membrane-mediated extractivefermentation for lactic acid production from cellulosic biomass, Appl.Biochem. Biotechnol. 63-65: 435-448.

In another preferred aspect, the fermentation product is a ketone. Itwill be understood that the term “ketone” encompasses a substance thatcontains one or more ketone moieties. In another more preferred aspect,the ketone is acetone. See, for example, Qureshi and Blaschek, 2003,supra.

In another preferred aspect, the fermentation product is an amino acid.In another more preferred aspect, the organic acid is aspartic acid. Inanother more preferred aspect, the amino acid is glutamic acid. Inanother more preferred aspect, the amino acid is glycine. In anothermore preferred aspect, the amino acid is lysine. In another morepreferred aspect, the amino acid is serine. In another more preferredaspect, the amino acid is threonine. See, for example, Richard andMargaritis, 2004, Empirical modeling of batch fermentation kinetics forpoly(glutamic acid) production and other microbial biopolymers,Biotechnology and Bioengineering 87(4): 501-515.

In another preferred aspect, the fermentation product is a gas. Inanother more preferred aspect, the gas is methane. In another morepreferred aspect, the gas is H₂. In another more preferred aspect, thegas is CO₂. In another more preferred aspect, the gas is CO. See, forexample, Kataoka et al., 1997, Studies on hydrogen production bycontinuous culture system of hydrogen-producing anaerobic bacteria,Water Science and Technology 36 (6-7): 41-47; and Gunaseelan, 1997,Biomass and Bioenergy 13(1-2): 83-114, Anaerobic digestion of biomassfor methane production: A review.

Recovery. The fermentation product(s) can be optionally recovered fromthe fermentation medium using any method known in the art including, butnot limited to, chromatography, electrophoretic procedures, differentialsolubility, distillation, or extraction. For example, alcohol isseparated from the fermented cellulosic material and purified byconventional methods of distillation. Ethanol with a purity of up toabout 96 vol. % can be obtained, which can be used as, for example, fuelethanol, drinking ethanol, i.e., potable neutral spirits, or industrialethanol.

Signal Peptide

The present invention also relates to an isolated polynucleotideencoding a signal peptide comprising or consisting of amino acids 1 to18 of SEQ ID NO: 2 or 1 to 25 of SEQ I DNO: 4. In one aspect, thepolynucleotide is nucleotides 1 to 54 of SEQ ID NO: 1 or 1 to 75 of SEQID NO: 3. The polynucleotides may further comprise a gene encoding aprotein, which is operably linked to the signal peptide.

The present invention also relates to nucleic acid constructs,expression vectors and recombinant host cells comprising suchpolynucleotides.

The present invention also relates to methods of producing a protein,comprising: (a) cultivating a recombinant host cell comprising such apolynucleotide operably linked to a gene encoding the protein; and (b)recovering the protein.

The protein may be native or heterologous to a host cell. The term“protein” is not meant herein to refer to a specific length of theencoded product and, therefore, encompasses peptides, oligopeptides, andpolypeptides. The term “protein” also encompasses two or morepolypeptides combined to form the encoded product. The proteins alsoinclude hybrid polypeptides and fused polypeptides.

Preferably, the protein is a hormone or variant thereof, enzyme,receptor or portion thereof, antibody or portion thereof, or reporter.For example, the protein may be an oxidoreductase, transferase,hydrolase, lyase, isomerase, or ligase such as an aminopeptidase,amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase,alpha-galactosidase, beta-galactosidase, glucoamylase,alpha-glucosidase, beta-glucosidase, invertase, laccase, another lipase,mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease,transglutaminase or xylanase.

The gene may be obtained from any prokaryotic, eukaryotic, or othersource.

List of Preferred Embodiments

-   Embodiment 1 An isolated polypeptide having cellobiohydrolase    activity selected from the group consisting of:

(a) a polypeptide having at least 90% sequence identity to the maturepolypeptide of SEQ ID NO: 2 or a polypeptide having at least 80%sequence identity to the mature polypeptide of SEQ ID NO: 4;

(b) a polypeptide encoded by a polynucleotide having at least 90%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 or the cDNA sequence thereof or a polypeptide encoded by apolynucleotide having at least 80% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 3 or the genomic DNA sequencethereof;

(c) a variant comprising a substitution, deletion, and/or insertion ofone or more (several) amino acids of the mature polypeptide of SEQ IDNO: 2 or of SEQ ID NO: 4; and

(d) a fragment of the polypeptide of (a), (b), or (c) that hascellobiohydrolase activity.

-   Embodiment 2. The polypeptide of embodiment 1, having at least 90%,    e.g. at least 92%, at least 95%, at least 96%, at least 97%, at    least 98%, at least 99%, or 100% sequence identity to the mature    polypeptide of SEQ ID NO: 2.-   Embodiment 3. The polypeptide of embodiment 1, having at least 80%,    e.g. at least 85%, at least 87%, at least 90%, at least 92%, at    least 95%, e.g., at least 96%, at least 97%, at least 98%, at least    99%, or 100% sequence identity to the mature polypeptide of SEQ ID    NO: 4.-   Embodiment 4. The polypeptides of any of embodiments 1-3, comprising    or consisting of SEQ ID NO: 2 or SEQ ID NO. 4.-   Embodiment 5. The polypeptides of embodiment 4, comprising or    consisting of the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO:    4.-   Embodiment 6. The polypeptides of embodiment 5, wherein the mature    polypeptide is amino acids 19 to 455 of SEQ ID NO: 2 or 26 to 537 of    SEQ ID NO: 4.-   Embodiment 7. An isolated polypeptide comprising a catalytic domain    selected from the group consisting of:

(a) a catalytic domain having at least 90% sequence identity to thecatalytic domain of SEQ ID NO: 2 or a catalytic domain having at least80% sequence identity to the catalytic domain of SEQ ID NO: 4;

(b) a catalytic domain encoded by a polynucleotide having at least 90%sequence identity to the catalytic domain coding sequence of SEQ ID NO:1 or a catalytic domain encoded by a polynucleotide having at least 80%sequence identity to the catalytic domain coding sequence of SEQ ID NO:3;

(c) a variant of a catalytic domain comprising a substitution, deletion,and/or insertion of one or more (several) amino acids of the catalyticdomain of SEQ ID NO: 2 or of SEQ ID NO: 4; and

(d) a fragment of a catalytic domain of (a), (b), or (c), which hascellobiohydrolase activity.

-   Embodiment 8. The polypeptide of embodiment 7, comprising or    consisting of the catalytic domain of SEQ ID NO: 2 or SEQ ID NO: 4.-   Embodiment 9. The polypeptide of embodiment 8, wherein the catalytic    domain is amino acids 19 to 455 of SEQ ID NO: 2 or 26 to 465 of SEQ    ID NO: 4.-   Embodiment 10. The polypeptide of any of embodiments 7-9, further    comprising a cellulose binding domain.-   Embodiment 11. A composition comprising the polypeptide of any of    embodiments 1-10.-   Embodiment 12. An isolated polynucleotide encoding the polypeptide    of any of embodiments 1-10.-   Embodiment 13. A nucleic acid construct or expression vector    comprising the polynucleotide of embodiment 12 operably linked to    one or more (several) control sequences that direct the production    of the polypeptide in an expression host.-   Embodiment 14. A recombinant host cell comprising the polynucleotide    of embodiment 12 operably linked to one or more control sequences    that direct the production of the polypeptide.-   Embodiment 15. A method of producing the polypeptide of any of    embodiments 1-10, comprising:

(a) cultivating a cell, which in its wild-type form produces thepolypeptide, under conditions conducive for production of thepolypeptide; and

(b) recovering the polypeptide.

-   Embodiment 16. A method of producing a polypeptide having    cellobiohydrolase activity, comprising:

(a) cultivating the recombinant host cell of embodiment 14 underconditions conducive for production of the polypeptide; and

(b) recovering the polypeptide.

-   Embodiment 17. A method for degrading or converting a cellulosic    material, comprising: treating the cellulosic material with an    enzyme composition in the presence of the polypeptide having    cellobiohydrolase activity of any of embodiments 1-10.-   Embodiment 18. The method of embodiment 17, wherein the cellulosic    material is pretreated.-   Embodiment 19. The method of embodiment 17 or 18, further comprising    recovering the degraded cellulosic material.-   Embodiment 20. The method of any of embodiments 17-19, wherein the    enzyme composition comprises one or more (several) enzymes selected    from the group consisting of a cellulase, a GH61 polypeptide having    cellulolytic enhancing activity, a hemicellulase, an expansin, an    esterase, a laccase, a ligninolytic enzyme, a pectinase, a    peroxidase, a protease, and a swollenin.-   Embodiment 21. The method of embodiment 20, wherein the cellulase is    one or more enzymes selected from the group consisting of an    endoglucanase, an additional cellobiohydrolase, and a    beta-glucosidase.-   Embodiment 22. The method of embodiment 21, wherein the    hemicellulase is one or more enzymes selected from the group    consisting of a xylanase, an acetyxylan esterase, a feruloyl    esterase, an arabinofuranosidase, a xylosidase, and a glucuronidase.-   Embodiment 23. A method for producing a fermentation product,    comprising:

(a) saccharifying a cellulosic material with an enzyme composition inthe presence of a polypeptide having endoglucanase activity of any ofembodiments 1-10;

(b) fermenting the saccharified cellulosic material with one or morefermenting microorganisms to produce the fermentation product; and

(c) recovering the fermentation product from the fermentation.

-   Embodiment 24. The method of embodiment 23, wherein the cellulosic    material is pretreated.-   Embodiment 25. The method of embodiment 23 or 24 wherein the enzyme    composition comprises one or more enzymes selected from the group    consisting of a cellulase, a GH61 polypeptide having cellulolytic    enhancing activity, a hemicellulase, an expansin, an esterase, a    laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a    protease, and a swollenin.-   Embodiment 26. The method of embodiment 25, wherein the cellulase is    one or more enzymes selected from the group consisting of an    endoglucanase, an additional cellobiohydrolase, and a    beta-glucosidase.-   Embodiment 27. The method of embodiment 25, wherein the    hemicellulase is one or more enzymes selected from the group    consisting of a xylanase, an acetyxylan esterase, a feruloyl    esterase, an arabinofuranosidase, a xylosidase, and a glucuronidase.-   Embodiment 28. The method of any of embodiments 23-27, wherein    steps (a) and (b) are performed simultaneously in a simultaneous    saccharification and fermentation.-   Embodiment 29. The method of any of embodiments 23-28, wherein the    fermentation product is an alcohol, an organic acid, a ketone, an    amino acid, or a gas.-   Embodiment 30. A method of fermenting a cellulosic material,    comprising: fermenting the cellulosic material with one or more    fermenting microorganisms, wherein the cellulosic material is    saccharified with an enzyme composition in the presence of a    polypeptide having cellobiohydrolase activity of any of embodiments    1-10.-   Embodiment 31. The method of embodiment 30, wherein the cellulosic    material is pretreated before saccharification.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES

Strains

Talaromyces byssochlamydoides strain CBS413.71 was used as the source offamily GH7 genes.

Aspergillus oryzae MT3568 strain was used for heterologous expression ofthe family GH7 genes encoding polypeptide having homology withpolypeptides with cellobiohydrolase activity. A. oryzae MT3568 is anamdS (acetamidase) disrupted gene derivative of Aspergillus oryzaeJaL355 (WO 2002/40694) in which pyrG auxotrophy was restored bydisrupting the A. oryzae acetamidase (amdS) gene with the pyrG gene.

Media

YP+2% glucose medium was composed of 1% yeast extract, 2% peptone and 2%glucose.

PDA agar plates were composed of potato infusion (potato infusion wasmade by boiling 300 g of sliced (washed but unpeeled) potatoes in waterfor 30 minutes and then decanting or straining the broth throughcheesecloth. Distilled water was then added until the total volume ofthe suspension was one liter, followed by 20 g of dextrose and 20 g ofagar powder. The medium was sterilized by autoclaving at 15 psi for 15minutes (Bacteriological Analytical Manual, 8th Edition, Revision A,1998).

LB plates were composed of 10 g of Bacto-Tryptone, 5 g of yeast extract,10 g of sodium chloride, 15 g of Bacto-agar, and deionized water to 1liter.

LB medium was composed of 10 g of Bacto-Tryptone, 5 g of yeast extract,and 10 g of sodium chloride, and deionized water to 1 liter.

COVE sucrose plates were composed of 342 g of sucrose, 20 g of agarpowder, 20 ml of COVE salt solution, and deionized water to 1 liter. Themedium was sterilized by autoclaving at 15 psi for 15 minutes(Bacteriological Analytical Manual, 8th Edition, Revision A, 1998). Themedium was cooled to 60° C. and 10 mM acetamide, 15 mM CsCl, TritonX-100 (50 μl/500 ml) were added.

COVE salt solution was composed of 26 g of MgSO₄.7H₂O, 26 g of KCL, 26 gof KH₂PO₄, 50 ml of COVE trace metal solution, and deionized water to 1liter.

COVE trace metal solution was composed of 0.04 g of Na₂B₄O₇.10H₂O, 0.4 gof CuSO₄.5H₂O, 1.2 g of FeSO₄.7H₂O, 0.7 g of MnSO₄. H₂O, 0.8 g ofNa₂MoO₄.2H₂O, 10 g of ZnSO₄.7H₂O, and deionized water to 1 liter.

Example 1 Cloning of Two Family GH7 Genes from TalaromycesByssochiamydoides

A set of degenerate primers shown below (SEQ ID NO: 5 to 10) weredesigned according to the strategy described by Rose et al., 1998,Nucleic Acids Research 26: 1628-1635, to target genes encodingcellobiohydrolase belonging to family GH7.

For1 SEQ ID NO: 5 GTCGTTCTTGATGCgaaytggmgntgg For2 SEQ ID NO: 6CCTGCGCCCAGAACtgygcnbtnga For3 SEQ ID NO: 7TGACGTCGATGTCTCCAATytnccntgygg Rev1 SEQ ID NO: 8GCATCCGTCGGGTAGTCGswrtcnarcca Rev2 SEQ ID NO: 9GTCGGGTAGTCGGAATCCarccanwncat Rev3 SEQ ID NO: 10GCCTGGCGTGTCGcanggrtgnggThe upper case nucleotides represent the consensus clamp of the primerand the lower case nucleotides represent the degenerate core of theoligonucleotides (Rose et al., 1998, Nucleic Acids Research 26:1628-1635).

PCR screening was performed using two successive PCRs. Nine PCRreactions were carried out with the primer couples For1/Rev1, For1/Rev2,For2/Rev1, For2/Rev2, For3/Rev1, For3/Rev2, For1/Rev3, For2/Rev3, andFor3/Rev3. The Forward primers (0.33 μl of a 10 μM stock solution) werecombined with their corresponding reverse primers (0.33 μl of a 10 μMstock solution) in a 10 μl mixture containing 0.33 μl genomic DNA and 5μl of REDDYMIX™ Extensor PCR Master Mix 1 (ABgene Ltd., Surrey, UnitedKingdom). Genomic DNA was obtained from fresh mycelium of Talaromycesbyssochlamydoides (see strain part) grown on PDA (see media section)according to the procedure described in the FASTDNA® SPIN Kit(Q-BIOgene, Carlsbad, Calif., USA). The PCR reaction was performed usinga DYAD® Thermal Cycler (Bio-Rad Laboratories, Inc., Hercules, Calif.,USA) programmed for one cycle at 94° C. for 2 minutes; 9 cycles each at94° C. for 15 seconds, 68° C. for 30 seconds with a decrease of 1° C.for each cycle, and 68° C. for 1 minute 45 seconds; 24 cycles each at94° C. for 15 seconds, 68° C. for 30 seconds, and 68° C. for 1 minute 45seconds; and extension at 68° C. for 7 minutes.

PCR products obtained during the first PCR were re-amplified with theircorresponding primers by transferring 0.5 μl of the first PCR reactionto a second 20 μl mixture containing the same concentration of primers,dNTPs, DNA polymerase, and buffer. The second PCR was performed using aDYAD® Thermal Cycler programmed for one cycle at 94° C. for 2 minutes;34 cycles each at 94° C. for 15 seconds, 58° C. for 30 seconds, and 68°C. for 1 minute 45 seconds; and a final extension at 68° C. for 7minutes.

PCR products obtained during the second amplification were analyzed by1% agarose gel electrophoresis using 40 mM Tris base-20 mM sodiumacetate-1 mM disodium EDTA (TAE) buffer. Single bands ranging from 200to 1200 nucleotides in size were excised from the gel and purified usinga GFX® PCR DNA and Gel Band Purification Kit (GE Healthcare, HiHerodDenmark) according to the manufacturer's instructions. Purified DNAsamples were directly sequenced with primers used for amplification.Sequences were assembled by SeqMan v7.2.1 (DNA star, Madison, Wis., USA)into contigs that were used as templates for designing GeneWalkingprimers shown below (SEQ ID NO: 11 to 22), following the constraintsdescribed in the GENE WALKING SPEEDUP™ Kit protocol (Seegene, Inc.,Seoul, Korea).

5220CBH1-1TSP1f SEQ ID NO: 11 AACAACTTTGATACACACGGCGG 5220CBH1-1TSP2fSEQ ID NO: 12 CTGCAGCAGGGTATGGTTCTGG 5220CBH1-1TSP3f SEQ ID NO: 13TGGTGATGAGTCTGTGGGACGG 5220CBH1-1TSP1r SEQ ID NO: 14GTCAGCAGCCATGGTAACAAGG 5220CBH1-1TSP2r SEQ ID NO: 15TGGTAACAAGGTACAGAGCGCCG 5220CBH1-1TSP3r SEQ ID NO: 16GTACAGAGCGCCGTTTAATCCGC 5220CBH1-2TSP1f SEQ ID NO: 17ACTGTACCGCTGAGAATTCTGTC 5220CBH1-2TSP2f SEQ ID NO: 18GCATGAGTGGTGTCAGTGAGGC 5220CBH1-2TSP3f SEQ ID NO: 19TGGTGTCAGTGAGGCTCTGTCC 5220CBH1-2TSP1r SEQ ID NO: 20CCATAACAACGAGGTAGAGGGC 5220CBH1-2TSP2r SEQ ID NO: 21CAGGGCAGGTTTGAGACATCCAC 5220CBH1-2TSP3r SEQ ID NO: 22TCTGGTAGTGGGTGTCATCCGC

GeneWalking was based on the protocol from the GENE WALKING SPEEDUP™ Kitwith some minor changes. Three PCR amplifications were carried out andin all cases, the REDDYMIX™ Extensor PCR Master Mix 1 (ABgene Ltd.,Surrey, United Kingdom) was used instead of the PCR enzyme mix presentin the Kit. GeneWalking PCR step 1 was performed in a total volume of 15μl by mixing 1.2 μl of primer 1 to 4 (2.5 μM) from the GENE WALKINGSPEEDUP™ Kit and 0.3 μl of the primers SEQ ID NO: 11 or SEQ ID NO: 14(10 μM) in the presence of 7.5 μl of REDDYMIX™ Extensor PCR Master Mix1, and 0.4 μl of T. byssochlamydoides genomic DNA. The PCR was performedusing a DYAD® Thermal Cycler programmed for one cycle at 94° C. for 3minutes followed by 1 minute at 42° C. and 2 minutes at 68° C.; 30cycles each at 94° C. for 30 seconds, 58° C. for 30 seconds, and 68° C.for 1 minute and 40 seconds; and elongation at 68° C. for 7 minutes. A0.5 μl aliquot of the amplification reaction was transferred to a secondPCR tube containing a 20 μl mixture composed of 10 μl of REDDYMIX™Extensor PCR Master Mix 1, 1 μl of primer 5 (10 μM) from the Kit, 1 μlof primers SEQ ID NO: 12 or SEQ ID NO: 15 (10 μM). The amplification wasperformed in a DYAD® Thermal Cycler programmed for denaturation at 94°C. for 3 minutes; 35 cycles each at 94° C. for 30 seconds, 58° C. for 30seconds, and 68° C. for 1 minute and 40 seconds; and elongation at 68°C. for 7 minutes. A 0.5 μl aliquot of the second amplification reactionwas transferred to a third PCR tube containing a 20 μl mixture composedof 10 μl of REDDYMIX™ Extensor PCR Master Mix 1, 1 μl of primer 6 (10μM) from the Kit, 1 μl of primers SEQ ID NO: 13 or SEQ ID NO: 16 (10μM). The amplification was performed in a DYAD® Thermal Cyclerprogrammed for denaturation at 94° C. for 3 minutes; 35 cycles each at94° C. for 30 seconds, 58° C. for 30 seconds, and 68° C. for 1 minuteand 40 seconds; and elongation at 68° C. for 7 minutes.

Similarly three successive PCR reactions were carried out in order toidentify the 5′ end and the 3′ end of the second gene from Talaromycesbyssochlamydoides, using the primers SEQ ID NO: 17 to 22.

The PCR products were directly sequenced using the two specific primersused for the last PCR. Resulting sequence chromatographs were assembledby SeqMan v7.2.1 (DNA star, Madison, Wis., USA) and resulting contigswere analyzed by blastx against protein databases, including Uniprot.The different contigs were analyzed for their identities to proteinsbelonging to GH7 family. Based on blastx analyses, the start codon ofthe genes were identified and the primers shown below were designed forcloning the genes into the expression vector pDAu109 (WO 2005/042735)using an IN-FUSION™′ Dry-Down PCR Cloning Kit (BD Biosciences, PaloAlto, Calif., USA).

5220CBHI1F (SEQ ID NO: 23)ACACAACTGGGGATCCACCATGTTTCGACGGGCTCTTTTCCTGTCC 5220CBHI2F(SEQ ID NO: 24) ACACAACTGGGGATCCACCATGTCCGCCTCTCTTTCTTACAGACTCTACG

Similarly, the result of the blastx analyse for the 3′ end of the genesidentify one stop codon for each of the two family GH7 genes identifiedin Talaromyces byssochlamydoides. Reverse primers shown below weredesigned for cloning the genes into the expression vector pDAu109 (WO2005/042735) using an IN-FUSION™ Dry-Down PCR Cloning Kit (BDBiosciences, Palo Alto, Calif., USA).

5220CBHI1R (SEQ ID NO: 25) AGATCTCGAGAAGCTTACGAAGTGGTGAAGGTCGAGTTGATTG5220CBHI2R (SEQ ID NO: 26) AGATCTCGAGAAGCTTACAGACACTGGGAGTAGTAAGGGTTC

The two cellobiohydrolase genes were amplified by PCR using the forwardand reverse cloning primers described above (SEQ ID NO 23 to 26) with T.byssochlamydoides strain CBS413.71 genomic DNA. The PCR was composed of1 μl of genomic DNA, 2.5 μl of cloning primer forward (10 μM), 2.5 μl ofprimer cloning primer reverse (10 μM), 10 μl of 5×HF buffer (FinnzymesOy, Finland), 1.6 μl of 50 mM MgCl₂, 2 μl of 10 mM dNTP, 0.5 μl ofPHUSION® DNA polymerase (Finnzymes Oy, Finland), and PCR-grade water to50 μl. The amplification reaction was performed using a DYAD® ThermalCycler programmed for 2 minutes at 98° C. followed by 19 touchdowncycles each at 98° C. for 15 seconds, 70° C. (−1° C./cycle) for 30seconds, and 72° C. for 2 minutes and 30 seconds; and 25 cycles each at98° C. for 15 seconds, 60° C. for 30 seconds, 72° C. for 2 minutes and30 seconds, and 5 minutes at 72° C.

The PCR products were isolated on 1.0% agarose gel electrophoresis usingTAE buffer where approximately 1.5 to 1.6 kb PCR bands were excised fromthe gel and purified using a GFX® PCR DNA and Gel Band Purification Kit(GE Healthcare, Hillerød Denmark) according to manufacturer'sinstructions. Fragments corresponding to the Talaromycesbyssochlamydoides and cellobiohydrolase genes were cloned into theexpression vector pDAu109 (WO 2005/042735) previously linearized withBam HI and Hind III, using an IN-FUSION™ Dry-Down PCR Cloning Kit (BDBiosciences, Palo Alto, Calif., USA) according to the manufacturer'sinstructions.

A 2.5 μl volume of the diluted ligation mixture was used to transform E.coli TOP10 chemically competent cells (Invitrogen, Carlsbad, Calif.,USA). Three colonies were selected on LB agar plates containing 100 μgof ampicillin per ml and cultivated overnight in 3 ml of LB mediumsupplemented with 100 μg of ampicillin per ml. Plasmid DNA was purifiedusing an E.Z.N.A.® Plasmid Mini Kit (Omega Bio-Tek, Inc., Norcross, Ga.,USA) according to the manufacturer's instructions. The two Talaromycesbyssochlamydoides cellobiohydrolase gene sequences were verified bySanger sequencing before heterologous expression. Two plasmidsdesignated as IF317#1 (containing gene SEQ ID NO: 1), and IF314#1(containing gene SEQ ID NO: 3) were selected for heterologous expressionof their cellobiohydrolase in an Aspergillus oryzae host cell.

Example 2 Characterization of the Talaromyces Byssochiamydoides GenomicDNAs Encoding Family GH7 Polypeptides

The genomic DNA sequence (SEQ ID NO: 1) and deduced amino acid sequence(SEQ ID NO: 2) of the Talaromyces byssochlamydoides GH7 gene is in thesequence list. The first (SEQ ID NO: 1) coding sequence is 1507 bpincluding the stop codon with 2 predicted introns (604 to 667 and 1236to 1310). The encoded predicted protein is 455 amino acids. Using theSignalP program version 3 (Nielsen et al., 1997, Protein Engineering 10:1-6), a signal peptide of 18 residues was predicted. The predictedcatalytic domain was identified from position 19 to 455 as defined bythe CaZy group (www.cazy.org). The predicted mature protein contains 437amino acids with a predicted molecular mass of 46.4 kDa and anisoelectric point of 3.9.

A comparative pairwise global alignment of amino acid sequence (SEQ IDNO: 2) was determined using the Needleman and Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) with gap openpenalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 matrix.The alignment showed that the deduced amino acid sequence of the T.byssochlamydoides cDNA encoding a family GH7 polypeptide having homologyto proteins with cellobiohydrolase activity shares 87.41% identity(excluding gaps) to the deduced amino acid sequence of ancellobiohydrolase from Talaromyces emersonii (GENESEQP: AYL28232).

The genomic DNA sequence (SEQ ID NO: 3) and deduced amino acid sequence(SEQ ID NO: 4) of the Talaromyces byssochlamydoides GH7 gene is in thesequence list. The second (SEQ ID NO: 3) coding sequence is 1614 bpincluding the stop codon. The encoded predicted protein is 537 aminoacids. Using the SignalP program (Nielsen et al., 1997, ProteinEngineering 10: 1-6), a signal peptide of 25 residues was predicted.Different domains (as defined by the CaZy group (www.cazy.org)) are alsopredicted on the protein: the catalytical domain positions 26 to 465 ofSEQ ID NO: 4, a linker positions 452 to 495 of SEQ ID NO: 4, and acellulose binding motif (CBM1) from positions 502 to 537 of SEQ ID NO:4. The predicted mature protein contains 512 amino acids with apredicted molecular mass of 53.5 kDa and an isoelectric point of 3.7.

A comparative pairwise global alignment of amino acid sequence (SEQ IDNO: 4) was determined using the Needleman and Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) with gap openpenalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 matrix.The alignment showed that the deduced amino acid sequence of the T.byssochlamydoides cDNA encoding a family GH7 polypeptide having homologyto proteins with cellobiohydrolase activity shares 78.94% identity(excluding gaps) to the deduced amino acid sequence of an glycosylhydrolase family 7 protein from Neosartorya fischeri (SWISSPROT:A1DAP8).

Example 3 Transformation of Aspergillus oryzae with Genes EncodingCellobiohydrolases from Talaromyces Byssochlamydoides

Protoplasts of Aspergillus oryzae MT3568 were prepared according to WO95/002043. One hundred μl of protoplasts were mixed with 2.5-15 μg ofthe Aspergillus expression vectors IF317#1, and IF314#1 (Example 1) and250 μl of 60% PEG 4000 (Applichem, Darmstadt, Germany) (polyethyleneglycol, molecular weight 4,000), 10 mM CaCl₂, and 10 mM Tris-HCl pH 7.5and gently mixed. The mixture was incubated at 37° C. for 30 minutes andthe protoplasts were spread onto COVE plates for selection. Afterincubation for 4-7 days at 37° C. spores of eight transformants wereinoculated into 0.5 ml of YP medium supplemented with 2% maltodextrin in96 deep well plates. After 4 days cultivation at 30° C., the culturebroths were analyzed by SDS-PAGE to identify the transformants producingthe largest amount of recombinant cellobiohydrolases from Talaromycesbyssochlamydoides.

Spores of the best transformant were spread on COVE plates containing0.01% TRITON® X-100 in order to isolate single colonies. The spreadingwas repeated twice in total on COVE plates containing 10 mM sodiumnitrate.

Example 4 Purification of Talaromyces Byssochlamydoides GH7Cellobiohydrolase I

The Aspergillus oryzae broth containing Talaromyces byssochlamydoidesGH7 Cellobiohydrolase I (P247B5, disclosed as SEQ ID NO: 2) was filteredusing a 0.22 μm EXPRESS™ Plus Membrane (Millipore, Bedford, Mass., USA).Filtered broth was concentrated and buffer exchanged using a Vivacell100 spin concentrator equipped with a 10 kDa polyethersulfone membrane(Sartorius Stedim Biotech S. A., Aubagne Cedex, France) with 20 mMTris-HCl pH 8.5 and then purified over a MONOQ™ HR 16/10 ion exchangechromatography column (GE Healthcare, Piscataway, N.J., USA) in 20 mMTris-HCl pH 8.5, over a linear 0 to 0.6 M NaCl gradient. Fractionscontaining the cellobiohydrolase I were pooled based on 8-16% CRITERION®Stain-free SDS-PAGE (Bio-Rad Laboratories, Inc., Hercules, Calif., USA).Protein concentration was determined using a Microplate BCA™ ProteinAssay Kit (Thermo Fischer Scientific, Waltham, Mass., USA) in whichbovine serum albumin was used as a protein standard.

Example 5 Pretreated Corn Stover Hydrolysis Assay

Corn stover was pretreated at the U.S. Department of Energy NationalRenewable Energy Laboratory (NREL) using 1.4 wt % sulfuric acid at 165°C. and 107 psi for 8 minutes. The water-insoluble solids in thepretreated corn stover (PCS) contained 56.5% cellulose, 4.6%hemicelluloses, and 28.4% lignin. Cellulose and hemicellulose weredetermined by a two-stage sulfuric acid hydrolysis with subsequentanalysis of sugars by high performance liquid chromatography using NRELStandard Analytical Procedure #002. Lignin was determinedgravimetrically after hydrolyzing the cellulose and hemicellulosefractions with sulfuric acid using NREL Standard Analytical Procedure#003.

Milled unwashed PCS (dry weight 32.35%) was prepared by milling wholeslurry PCS in a Cosmos ICMG 40 wet multi-utility grinder (EssEmmCorporation, Tamil Nadu, India).

The hydrolysis of PCS was conducted using 2.2 ml deep-well plates(Axygen, Union City, Calif., USA) in a total reaction volume of 1.0 ml.The hydrolysis was performed with 50 mg of insoluble PCS solids per mlof 50 mM sodium acetate pH 5.0 buffer containing 1 mM manganese sulfateand various protein loadings of various enzyme compositions (expressedas mg protein per gram of cellulose). Enzyme compositions were preparedas described in Example 6, and then added simultaneously to all wells ina volume ranging from 50 μl to 200 μl, for a final volume of 1 ml ineach reaction. The plates were then sealed using an ALPS300™ plate heatsealer (Abgene, Epsom, United Kingdom), mixed thoroughly, and incubatedat a specific temperature for 72 hours. All experiments reported wereperformed in triplicate.

Following hydrolysis, samples were filtered using a 0.45 μm MULTISCREEN®96-well filter plate (Millipore, Bedford, Mass., USA) and filtratesanalyzed for sugar content as described below. When not usedimmediately, filtered aliquots were frozen at −20° C. The sugarconcentrations of samples diluted in 0.005 M H₂SO₄ were measured using a4.6×250 mm AMINEX® HPX-87H column (Bio-Rad Laboratories, Inc., Hercules,Calif., USA) by elution with 0.05% w/w benzoic acid-0.005 M H₂SO₄ at 65°C. at a flow rate of 0.6 ml per minute, and quantitation by integrationof the glucose, cellobiose, and xylose signals from refractive indexdetection (CHEMSTATION®, AGILENT® 1100 HPLC, Agilent Technologies, SantaClara, Calif., USA) calibrated by pure sugar samples. The resultantglucose and cellobiose equivalents were used to calculate the percentageof cellulose conversion for each reaction.

Glucose, cellobiose, and xylose were measured individually. Measuredsugar concentrations were adjusted for the appropriate dilution factor.In case of unwashed PCS, the net concentrations ofenzymatically-produced sugars were determined by adjusting the measuredsugar concentrations for corresponding background sugar concentrationsin unwashed PCS at zero time points. All HPLC data processing wasperformed using MICROSOFT EXCEL™ software (Microsoft, Richland, Wash.,USA)

The degree of cellulose conversion to glucose was calculated using thefollowing equation: % conversion=(glucose concentration/glucoseconcentration in a limit digest)×100. In order to calculate %conversion, a 100% conversion point was set based on a cellulase control(100 mg of Trichoderma reesei cellulase per gram cellulose), and allvalues were divided by this number and then multiplied by 100.Triplicate data points were averaged and standard deviation wascalculated.

Example 6 Preparation of High-temperature Enzyme Composition

Preparation of Aspergillus fumigatus cellobiohydrolase II. TheAspergillus fumigatus GH6A cellobiohydrolase II (SEQ ID NO: 18 in WO2011/057140) was prepared recombinantly in Aspergillus oryzae asdescribed in WO 2011/057140. The filtered broth of Aspergillus fumigatusGH6A cellobiohydrolase II was buffer exchanged into 20 mM Tris pH 8.0using a 400 ml SEPHADEX™ G-25 column (GE Healthcare, United Kingdom)according to the manufacturer's instructions. The fractions were pooledand adjusted to 1.2 M ammonium sulphate-20 mM Tris pH 8.0. Theequilibrated protein was loaded onto a PHENYL SEPHAROSE™ 6 Fast Flowcolumn (high sub) (GE Healthcare, Piscataway, N.J., USA) equilibrated in20 mM Tris pH 8.0 with 1.2 M ammonium sulphate, and bound proteins wereeluted with 20 mM Tris pH 8.0 with no ammonium sulphate. The fractionswere pooled.

Preparation of Penicillium sp. (emersonii) GH61A polypeptide havingcellulolytic enhancing activity. The Penicillium sp. (emersonii) GH61Apolypeptide (SEQ ID NO: 2 in WO 2011/041397) was recombinantly preparedand purified according to WO 2011/041397.

Preparation of Trichoderma reesei GH5 endoglucanase II. The Trichodermareesei GH5 endoglucanase II (SEQ ID NO: 22 in WO 2011/057140) wasprepared recombinantly according to WO 2011/057140. The filtered brothof Trichoderma reesei GH5 endoglucanase II was desalted andbuffer-exchanged into 20 mM Tris pH 8.0 using tangential flow (10Kmembrane, Pall Corporation) according to the manufacturer'sinstructions.

Preparation of Aspergillus fumigatus Cel3A beta-glucosidase. Aspergillusfumigatus Cel3A beta-glucosidase (SEQ ID NO: 2 in WO 2005/047499) wasrecombinantly prepared according to WO 2005/047499 using Aspergillusoryzae as a host. The filtered broth of Aspergillus fumigatus Cel3Abeta-glucosidase was concentrated and buffer exchanged using atangential flow concentrator equipped with a 10 kDa polyethersulfonemembrane with 20 mM Tris-HCl pH 8.5. The sample was loaded onto a QSEPHAROSE® High Performance column (GE Healthcare, Piscataway, N.J.,USA) equilibrated in 20 mM Tris pH 8.5, and bound proteins were elutedwith a linear gradient from 0-600 mM sodium chloride. The fractions wereconcentrated and loaded onto a SUPERDEX® 75 HR 26/60 column equilibratedwith 20 mM Tris-150 mM sodium chloride pH 8.5.

Preparation of Aspergillus fumigatus GH10 xylanase. The Aspergillusfumigatus GH10 xylanase (xyn3) (SEQ ID NO: 48 in WO 2011/057140) wasprepared recombinantly according to WO 2006/078256 using Aspergillusoryzae BECh2 (WO 2000/39322) as a host. The filtered broth ofAspergillus fumigatus NN055679 GH10 xylanase (xyn3) was desalted andbuffer-exchanged into 50 mM sodium acetate pH 5.0 using a HIPREP® 26/10Desalting Column according to the manufacturer's instructions.

Preparation of Talaromyces emersonii GH3 beta-xylosidase. TheTalaromyces emersonii GH3 beta-xylosidase (SEQ ID NO: 60 in WO2011/057140) was prepared recombinantly in Aspergillus oryzae asdescribed in WO 2011/057140. The Talaromyces emersonii GH3beta-xylosidase was desalted and buffer-exchanged into 50 mM sodiumacetate pH 5.0 using tangential flow (10K membrane, Pall Corporation)according to the manufacturer's instructions.

The protein concentration for each of the monocomponents described abovewas determined using a Microplate BCA™ Protein Assay Kit in which bovineserum albumin was used as a protein standard. A high-temperature enzymecomposition was composed of each monocomponent, prepared as describedabove, as follows: 25% Aspergillus fumigatus Cel6A cellobiohydrolase II,15% Penicillium emersonii GH61A polypeptide having cellulolyticenhancing activity, 10% Trichoderma reesei GH5 endoglucanase II, 5%Aspergillus fumigatus GH10 xylanase (xyn3), 5% Aspergillus fumigatusbeta-glucosidase mutant, and 3% Talaromyces emersonii beta-xylosidase.The high-temperature enzyme composition is designated herein as“high-temperature enzyme composition without cellobiohydrolase I”.

Example 7 Effect of Talaromyces Byssochlamydoides GH7 CellobiohydrolaseI (SEQ ID NO: 2, P247B5) on a High-Temperature Enzyme Composition UsingMilled Unwashed PCS at 50-65° C.

The Talaromyces byssochlamydoides GH7 cellobiohydrolase I (P247B5) wasevaluated in a high-temperature enzyme composition withoutcellobiohydrolase I at 50° C., 55° C., 60° C., and 65° C. using milledunwashed PCS as a substrate. The high-temperature enzyme compositionwithout cellobiohydrolase I (Example 6) was added to PCS hydrolysisreactions at 1.9 mg total protein per g cellulose and 3.0 mg totalprotein per g cellulose, and the hydrolysis results were compared withthe results for a similar high-temperature enzyme composition with addedGH7 cellobiohydrolase I (3.0 mg protein per g cellulose).

The assay was performed as described in Example 5. The 1 ml reactionswith milled unwashed PCS (5% insoluble solids) were conducted for 72hours in 50 mM sodium acetate pH 5.0 buffer containing 1 mM manganesesulfate. All reactions were performed in triplicate and involved singlemixing at the beginning of hydrolysis.

The results shown in Table 1 demonstrated that at 50° C., 55° C., 60°C., and 65° C. the high-temperature enzyme composition that includedTalaromyces byssochlamydoides GH7 cellobiohydrolase I (P247B5)significantly outperformed the enzyme composition containing nocellobiohydrolase I.

TABLE 1 % Conversion (cellulose to glucose) Temperature ° C. Composition50 55 60 65 HT composition w/o CBHI (1.9 mg/g) 29.75 25.65 18.35 13.451HT composition w/o CBHI (3.0 mg/g) 37.06 31.72 22.01 17.572 HTcomposition with 37% 53.36 51.45 42.24 34.479 T. byssochlamydoides GH7CBHI (3.0 mg/g)

The invention claimed is:
 1. A method for degrading a cellulosicmaterial, comprising: treating the cellulosic material with an enzymecomposition comprising a polypeptide having cellobiohydrolase activityto produce a degraded cellulosic material and recovering the degradedcellulosic material, wherein the polypeptide having cellobiohydrolaseactivity is selected from the group consisting of: (a) a polypeptidecomprising an amino acid sequence having at least 90% sequence identityto the amino acid sequence of amino acids 26 to 537 of SEQ ID NO: 4; (b)a polypeptide encoded by a polynucleotide comprising a nucleotidesequence having at least 90% sequence identity to the nucleotidesequence of nucleotides 76 to 1614 of SEQ ID NO: 3; (c) a fragment ofthe polypeptide of (a) or (b), wherein the fragment hascellobiohydrolase activity; (d) a polypeptide comprising an amino acidsequence having at least 90% sequence identity to the amino acidsequence of amino acids 26 to 465 of SEQ ID NO: 4; (e) a polypeptideencoded by a polynucleotide comprising a nucleotide sequence having atleast 90% sequence identity to the nucleotide sequence of nucleotides 76to 1395 of SEQ ID NO: 3; and (f) a fragment of the polypeptide of (d) or(e), wherein the fragment has cellobiohydrolase activity.
 2. The methodof claim 1, wherein the cellulosic material is pretreated.
 3. The methodof claim 2, wherein the degraded cellulosic material is a sugar.
 4. Themethod of claim 3, wherein the sugar is glucose, cellobiose, xylose,xylulose, arabinose, mannose, galactose, and/or solubleoligosaccharides.
 5. The method of claim 1, wherein the enzymecomposition further comprises one or more enzymes selected from thegroup consisting of a cellulase, a glycoside hydrolase family 61 (GH61)polypeptide having cellulolytic enhancing activity, a hemicellulase, anexpansin, an esterase, a laccase, a ligninolytic enzyme, a pectinase, aperoxidase, a protease, and a swollenin.
 6. The method of claim 1,wherein the polypeptide having cellobiohydrolase activity comprises anamino acid sequence having at least 92% sequence identity to the aminoacid sequence of amino acids 26 to 537 of SEQ ID NO:
 4. 7. The method ofclaim 1, wherein the polypeptide having cellobiohydrolase activitycomprises the amino acid sequence of SEQ ID NO:
 4. 8. The method ofclaim 1, wherein the polypeptide having cellobiohydrolase activitycomprises the amino acid sequence of amino acids 26 to 537 of SEQ ID NO:4.
 9. The method of claim 1, wherein the polypeptide havingcellobiohydrolase activity comprises an amino acid sequence having atleast 92% sequence identity to the amino acid sequence of amino acids 26to 465 of SEQ ID NO:
 4. 10. The method of claim 1, wherein thepolypeptide having cellobiohydrolase activity comprises the amino acidsequence of amino acids 26 to 465 of SEQ ID NO:
 4. 11. The method ofclaim 10, wherein the polypeptide having cellobiohydrolase activityfurther comprises a cellulose binding domain.
 12. A method for producinga fermentation product, comprising: (a) saccharifying a cellulosicmaterial with an enzyme composition comprising a polypeptide havingcellobiohydrolase activity to produce a saccharified cellulosicmaterial, wherein the polypeptide having cellobiohydrolase activity isselected from the group consisting of: (i) a polypeptide comprising anamino acid sequence having at least 90% sequence identity to the aminoacid sequence of amino acids 26 to 537 of SEQ ID NO: 4; (ii) apolypeptide encoded by a polynucleotide comprising a nucleotide sequencehaving at least 90% sequence identity to the nucleotide sequence ofnucleotides 76 to 1614 of SEQ ID NO: 3; (iii) a fragment of thepolypeptide of (i) or (ii), wherein the fragment has cellobiohydrolaseactivity; (iv) a polypeptide comprising an amino acid sequence having atleast 90% sequence identity to the amino acid sequence of amino acids 26to 465 of SEQ ID NO: 4; (v) a polypeptide encoded by a polynucleotidecomprising a nucleotide sequence having at least 90% sequence identityto the nucleotide sequence of nucleotides 76 to 1395 of SEQ ID NO: 3;and (vi) a fragment of the polypeptide of (iv) or (v), wherein thefragment has cellobiohydrolase activity; (b) fermenting the saccharifiedcellulosic material with one or more fermenting microorganisms toproduce the fermentation product; and (c) recovering the fermentationproduct from the fermentation.
 13. The method of claim 12, wherein thecellulosic material is pretreated.
 14. The method of claim 12, whereinsteps (a) and (b) are performed simultaneously in a simultaneoussaccharification and fermentation.
 15. The method of claim 12, whereinthe enzyme composition further comprises one or more enzymes selectedfrom the group consisting of a cellulase, a GH61 polypeptide havingcellulolytic enhancing activity, a hemicellulase, an expansin, anesterase, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, aprotease, and a swollenin.
 16. The method of claim 12, wherein thefermentation product is an alcohol, an amino acid, a ketone, an organicacid, or a gas.
 17. The method of claim 12, wherein the polypeptidehaving cellobiohydrolase activity comprises an amino acid sequencehaving at least 92% sequence identity to the amino acid sequence ofamino acids 26 to 537 of SEQ ID NO:
 4. 18. The method of claim 12,wherein the polypeptide having cellobiohydrolase activity comprises theamino acid sequence of SEQ ID NO:
 4. 19. The method of claim 12, whereinthe polypeptide having cellobiohydrolase activity comprises the aminoacid sequence of amino acids 26 to 537 of SEQ ID NO:
 4. 20. The methodof claim 12, wherein the polypeptide having cellobiohydrolase activitycomprises an amino acid sequence having at least 92% sequence identityto the amino acid sequence of amino acids 26 to 465 of SEQ ID NO:
 4. 21.The method of claim 12, wherein the polypeptide having cellobiohydrolaseactivity comprises the amino acid sequence of amino acids 26 to 465 ofSEQ ID NO:
 4. 22. The method of claim 21, wherein the polypeptide havingcellobiohydrolase activity further comprises a cellulose binding domain.23. The method of claim 1, wherein the polypeptide havingcellobiohydrolase activity comprises an amino acid sequence having atleast 95% sequence identity to the amino acid sequence of amino acids 26to 537 of SEQ ID NO:
 4. 24. The method of claim 1, wherein thepolypeptide having cellobiohydrolase activity comprises an amino acidsequence having at least 97% sequence identity to the amino acidsequence of amino acids 26 to 537 of SEQ ID NO:
 4. 25. The method ofclaim 1, wherein the polypeptide having cellobiohydrolase activitycomprises an amino acid sequence having at least 99% sequence identityto the amino acid sequence of amino acids 26 to 537 of SEQ ID NO:
 4. 26.The method of claim 1, wherein the polypeptide having cellobiohydrolaseactivity comprises an amino acid sequence having at least 95% sequenceidentity to the amino acid sequence of amino acids 26 to 465 of SEQ IDNO:
 4. 27. The method of claim 1, wherein the polypeptide havingcellobiohydrolase activity comprises an amino acid sequence having atleast 97% sequence identity to the amino acid sequence of amino acids 26to 465 of SEQ ID NO:
 4. 28. The method of claim 21, wherein thepolypeptide having cellobiohydrolase activity comprises an amino acidsequence having at least 99% sequence identity to the amino acidsequence of amino acids 26 to 465 of SEQ ID NO:
 4. 29. The method ofclaim 12, wherein the polypeptide having cellobiohydrolase activitycomprises an amino acid sequence having at least 95% sequence identityto the amino acid sequence of amino acids 26 to 537 of SEQ ID NO:
 4. 30.The method of claim 12, wherein the polypeptide having cellobiohydrolaseactivity comprises an amino acid sequence having at least 97% sequenceidentity to the amino acid sequence of amino acids 26 to 537 of SEQ IDNO:
 4. 31. The method of claim 12, wherein the polypeptide havingcellobiohydrolase activity comprises an amino acid sequence having atleast 99% sequence identity to the amino acid sequence of amino acids 26to 537 of SEQ ID NO:
 4. 32. The method of claim 12, wherein thepolypeptide having cellobiohydrolase activity comprises an amino acidsequence having at least 95% sequence identity to the amino acidsequence of amino acids 26 to 465 of SEQ ID NO:
 4. 33. The method ofclaim 12, wherein the polypeptide having cellobiohydrolase activitycomprises an amino acid sequence having at least 97% sequence identityto the amino acid sequence of amino acids 26 to 465 of SEQ ID NO:
 4. 34.The method of claim 12, wherein the polypeptide having cellobiohydrolaseactivity comprises an amino acid sequence having at least 99% sequenceidentity to the amino acid sequence of amino acids 26 to 465 of SEQ IDNO:
 4. 35. The method of claim 1, wherein the polypeptide havingcellobiohydrolase activity comprises an amino acid sequence having atleast 96% sequence identity to the amino acid sequence of amino acids 26to 537 of SEQ ID NO:
 4. 36. The method of claim 1, wherein thepolypeptide having cellobiohydrolase activity comprises an amino acidsequence having at least 98% sequence identity to the amino acidsequence of amino acids 26 to 537 of SEQ ID NO:
 4. 37. The method ofclaim 1, wherein the polypeptide having cellobiohydrolase activitycomprises an amino acid sequence having at least 96% sequence identityto the amino acid sequence of amino acids 26 to 465 of SEQ ID NO:
 4. 38.The method of claim 1, wherein the polypeptide having cellobiohydrolaseactivity comprises an amino acid sequence having at least 98% sequenceidentity to the amino acid sequence of amino acids 26 to 465 of SEQ IDNO:
 4. 39. The method of claim 12, wherein the polypeptide havingcellobiohydrolase activity comprises an amino acid sequence having atleast 96% sequence identity to the amino acid sequence of amino acids 26to 537 of SEQ ID NO:
 4. 40. The method of claim 12, wherein thepolypeptide having cellobiohydrolase activity comprises an amino acidsequence having at least 98% sequence identity to the amino acidsequence of amino acids 26 to 537 of SEQ ID NO:
 4. 41. The method ofclaim 12, wherein the polypeptide having cellobiohydrolase activitycomprises an amino acid sequence having at least 96% sequence identityto the amino acid sequence of amino acids 26 to 465 of SEQ ID NO:
 4. 42.The method of claim 12, wherein the polypeptide having cellobiohydrolaseactivity comprises an amino acid sequence having at least 98% sequenceidentity to the amino acid sequence of amino acids 26 to 465 of SEQ IDNO: 4.